Volume 123, Issue 2, Pages 586-598 (August 2002) Lipopolysaccharide preconditioning and cross-tolerance: The induction of protective mechanisms for rat intestinal ileus  Nicolas T. Schwarz, Britta Engel, Mark K. Eskandari, Jörg C. Kalff, Jennifer R. Grandis, Anthony J. Bauer  Gastroenterology  Volume 123, Issue 2, Pages 586-598 (August 2002) DOI: 10.1053/gast.2002.34777 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Transit histograms of the distribution of nonabsorbable FITC-labeled dextran along the small intestine and colon 2 hours after oral LPS administration (SB, small bowel; CEC, cecum; COL, colon). The distribution histogram shows a significant delay in transit of the FITC-labeled marker measured in animals 24 hours after LPS injection (GC = 6.53) compared with controls (GC = 9.05, N = 5 each). □, Control ACI rats; ■, LPS 24 hours. Gastroenterology 2002 123, 586-598DOI: (10.1053/gast.2002.34777) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Plots of the temporal recovery of in vitro circular muscle contractions after a single injection and in the continued presence of LPS (12.5 mg/kg). (A) Graph shows bethanechol-stimulated dose-response curves generated from jejunal circular muscle strips prepared from controls and from animals obtained 1, 2, 3, 5, and 7 days after a single injection of LPS. Bethanechol-stimulated smooth muscle contractility recorded from LPS-injected rats recovered to control levels 7 days after acute challenge. ■, SD rat control; ♦, 24 hours after LPS; ▵, 48 hours after LPS; ○, 3 days after LOPS; □, 6 days after LPS; ♢, 7 days after LPS. (B) Graph shows bethanechol-stimulated circular smooth muscle contractility in controls and in rats subjected to repeated daily LPS injections. Muscle contractility from animals repeatedly injected with LPS recovered functional activity after the 7th day of treatment. ■, SD rat control; ♦, 24 hours after LPS; ▵, 3 days after LPS; ○, 4 days after LPS; □, 5 days after LPS; ♢, 7 days after LPS. (C) The temporal recovery functions of in vitro circular smooth muscle contractility recorded with 100 μmol/L bethanechol for animals that received a single injection of LPS and animals that received daily continuous injections of LPS. Comparatively, the recovery was similar in the 2 groups of animals. ■, Recovery; ♢, preconditioning. Gastroenterology 2002 123, 586-598DOI: (10.1053/gast.2002.34777) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 The EMSA gel shows the activation of the (A) transcription factors NF-κB and (B) STATs within the isolated jejunal muscularis in naive tissue extracts and extracts after single or repeated LPS injections in the rat. (A) Four hours after acute LPS injection (18.2-fold) or IM (32.2-fold), a significant activation in NF-κB was observed over controls. In extracts from preconditioned animals repeatedly injected with LPS, 4 hours after the 7th injection or 4 hours after IM of preconditioned animals, NF-κB was activated only 18.6% and 75.8% compared with LPS and manipulation extracts of naive tissues, respectively. (B) The EMSA gel shows the effect of LPS preconditioning on STAT activation. Control STAT levels were low compared with the intense activation 4 hours after LPS injection of naive animals (37.2-fold). In contrast, STAT phosphorylation from muscularis protein extracts of preconditioned animals 4 hours after the 7th LPS injection showed only 12.1% of the activation seen in LPS-stimulated naive tissues. Gastroenterology 2002 123, 586-598DOI: (10.1053/gast.2002.34777) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 LPS induction of IL-6 and TNF-α mRNA in the jejunal muscularis and mucosa. (A) The plot delineates the time course of LPS-stimulated IL-6 mRNA up-regulation within the jejunal muscularis and mucosa, showing that IL-6 message was rapidly induced within the gut wall and remained elevated for up to 6 hours after LPS. •, Muscularis; ▴, mucosa. (B) The effect of preconditioning on IL-6 induction by LPS. A significant 12.2-fold induction of muscularis IL-6 mRNA was observed 4 hours after a single endotoxin treatment compared with control levels. In contrast, after LPS preconditioning, IL-6 mRNA was only induced in the muscularis to 18.8% of the levels induced by LPS in naive tissues. A similar preconditioning effect was observed in the mucosa, but the initial naive induction by LPS was not as dramatic compared with the muscularis. (C) The naive and preconditioned effect of LPS on TNF-α induction. LPS induced a significant 14.2-fold induction of muscularis TNF-α mRNA 4 hours after a single endotoxin treatment compared with control levels. In contrast, after LPS preconditioning, TNF-α mRNA was only induced in the muscularis to 37.2% of the levels induced by LPS in naive tissues. A preconditioning effect in the mucosa for TNF-α was difficult to discern. Gastroenterology 2002 123, 586-598DOI: (10.1053/gast.2002.34777) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 LPS induction of iNOS mRNA in the jejunal muscularis and mucosa. (A) The plot delineates the time course of LPS-stimulated iNOS mRNA up-regulation within the jejunal muscularis and mucosa showing that iNOS message was rapidly induced within the gut wall and remained elevated for up to 6 hours after LPS. ●, Muscularis; ▴, mucosa. (B) The effect of preconditioning on iNOS induction by LPS. A significant 19.2-fold induction of muscularis iNOS mRNA was observed 4 hours after a single endotoxin treatment compared with control levels. In contrast, after LPS preconditioning, iNOS mRNA was induced in the muscularis to 67.6% of the levels induced by LPS in naive tissues. A similar preconditioning effect was observed in the mucosa. LPS-induced mucosal iNOS in naive tissues was 17.4-fold and induction in preconditioned tissues was only 19.1% of that seen in acutely stimulated naive tissues. (C) The lower band represents the respective unchanged mRNA amounts for the housekeeping gene GAPDH. Gastroenterology 2002 123, 586-598DOI: (10.1053/gast.2002.34777) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Protective effect of LPS preconditioning on intestinal manipulation induced suppression of in vitro jejunal circular smooth muscle contractility. The open circles (○) in the plot show the typical suppression in bethanechol-stimulated jejunal circular smooth muscle contractility recorded in vitro from rats after intestinal manipulation compared with controls (●). The cross (+) symbols show the complete prevention of manipulation-induced muscle suppression in animals that were preconditioned with LPS for 7 days. Gastroenterology 2002 123, 586-598DOI: (10.1053/gast.2002.34777) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Preconditioning effect of LPS on IM-induced (A) TNF-α, (B) IL-6, (C) iNOS, and (D) COX-2 mRNA up-regulation within the jejunal muscularis and mucosa. For each mRNA, IM caused a significant up-regulation compared with control. Preconditioning of the animals with daily low doses of LPS caused significant molecular cross-tolerance within the intestinal muscularis in animals, who had received IM 24 hours after the 7th day of LPS pretreatment. A preconditioning cross-tolerance effect for each mRNA in the mucosa was difficult to discern. Gastroenterology 2002 123, 586-598DOI: (10.1053/gast.2002.34777) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Myeloperoxidase-positive leukocytes within Hanker-Yates–stained muscularis whole mounts (A) prepared from the jejunum obtained from a control and (B) 24 hours after jejunal manipulation. (C) Neutrophilic recruitment after IM is significantly blunted in animals that had received LPS preconditioning (original magnification, 200×). Gastroenterology 2002 123, 586-598DOI: (10.1053/gast.2002.34777) Copyright © 2002 American Gastroenterological Association Terms and Conditions