The Role of Leukotriene B4 in Clostridium difficile Toxin A--Induced Ileitis in Rats  Douglas C. McVey, Steven R. Vigna  Gastroenterology  Volume 128, Issue 5, Pages 1306-1316 (May 2005) DOI: 10.1053/j.gastro.2005.03.017 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Effects of LTB4 on ileal luminal fluid accumulation, MPO activity, and histopathology and inhibition of these effects by pretreatment with capsazepine. (A, B) Vehicle controls are LTB4(0)/CPZ(−); (N = 6). (A) Dose-dependent LTB4 stimulation of ileal luminal fluid accumulation and the inhibition of this effect by pretreatment with 10 μg of capsazepine administered intraluminally 30 minutes before LTB4. (B) Dose-dependent LTB4 stimulation of ileal MPO activity and the inhibition of this effect by pretreatment with 10 μg of capsazepine administered intraluminally 30 minutes before LTB4. (C) H&E-stained sections of rat ileal segments after they were treated intraluminally for 3 hours with vehicle (left panel), 3 μg of LTB4 (middle panel), or 3 μg of LTB4 after pretreatment with 10 μg of capsazepine 30 minutes before LTB4 administration (right panel). Scale bar, 100 μm. *P < .05 vs LTB4 (0)/CPZ(−); **P < .001 vs LTB4 (0)/CPZ(−); #P < .05 vs LTB4 (3 μg)/CPZ(−); ##P < .001 vs LTB4 (3 μg)/CPZ(−). Gastroenterology 2005 128, 1306-1316DOI: (10.1053/j.gastro.2005.03.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 H&E-stained sections of rat ileal segments after they were treated intraluminally for 3 hours with (A) vehicle, (B) 3 μg of LTB4, or (C) 3 μg of LTB4 after pretreatment with 1 μg of I-RTX 30 minutes before LTB4 administration. Scale bar, 100 μm. Gastroenterology 2005 128, 1306-1316DOI: (10.1053/j.gastro.2005.03.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Effects of 30 minutes of intraluminal pretreatment of ileal segments with the LTB4-receptor antagonist U75302 at doses of 3, 10, or 30 μg on LTB4-stimulated (3 μg) luminal fluid accumulation and MPO activity. When administered alone at the doses tested, U75302 had no effect on luminal fluid accumulation or MPO activity (N = 6). *P < .01 vs 3 μg LTB4(+)/U75302(0). Gastroenterology 2005 128, 1306-1316DOI: (10.1053/j.gastro.2005.03.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Effects of intraluminal capsazepine and L-733,060 pretreatment before LTB4 administration on SP release in the ileum shown by endogenous SP-mediated NK-1R endocytosis in myenteric plexus ganglionic cell bodies. Laser scanning confocal fluorescent micrographs of neurons immunostained for the NK-1R from ileal segments treated for 3 hours with (A) vehicle, (B) 3 μg LTB4, (C) 3 μg LTB4 after 30 minutes of pretreatment with 10 μg capsazepine before LTB4 administration, (D) 3 μg LTB4 after 10 minutes pretreatment intraperitoneally with 3 mg/kg L-733,060 before LTB4 administration, or (E) 3 μg LTB4 after 10 minutes of pretreatment intraperitoneally with 3 mg/kg L-733,061 before LTB4 administration. Pretreatment with capsazepine and L-733,060, but not L-733,061, inhibited LTB4-induced endogenous SP release/action. Scale bar, 5 μm. Gastroenterology 2005 128, 1306-1316DOI: (10.1053/j.gastro.2005.03.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Quantitation of LTB4-induced SP release determined by endogenous SP-mediated NK-1R endocytosis in myenteric plexus ganglionic cell bodies. The percentage of NK-1R-ir myenteric plexus neuronal cell bodies with >10 NK-1R-ir endosomes was determined in 10 NK-1R-ir cells per animal for each treatment as an index of endogenous SP release (N = 6). (A) Capsazepine (cpz) pretreatment inhibits LTB4-induced SP release. *P < .001 vs LTB4(−)/CPZ(−); #P < .001 vs LTB4 (+)/CPZ(−). (B) L733,060 pretreatment inhibits LTB4-induced SP release. *P < .001 vs LTB4(−)/L733,060(−); #P < .001 vs LTB4(+)/L733,060(−). (C) Pretreatment with the inactive enantiomer of L733,060, L733,061, does not inhibit LTB4-induced SP release. *P < .001 vs LTB4(−)/L733,061(−). Gastroenterology 2005 128, 1306-1316DOI: (10.1053/j.gastro.2005.03.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Effects of the specific NK-1R antagonist L-733,060 (A, B: 3 mg/kg intraperitoneally) and its inactive enantiomer L-733,061 (C, D: 3 mg/kg intraperitoneally) administered 10 minutes before LTB4 on LTB4-stimulated (3 μg intraluminally) (A, C) ileal luminal fluid accumulation and (B, D) MPO activity. The specific NK-1R antagonist, L-733,060, significantly inhibited both LTB4-stimulated ileal luminal fluid accumulation and MPO activity whereas its inactive enantiomer, L-733,061, did not. L-733,060 and L-733,061 had no effects when given alone (data not shown) (N = 6). *P < .05 vs LTB4(−)/L-733,060(−) or L-733,061(−); **P < .001 vs LTB4(−)/L-733,060(−) or L-733,061(−); #P < .001 vs LTB4(+)/L-733,060(−). Gastroenterology 2005 128, 1306-1316DOI: (10.1053/j.gastro.2005.03.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Effects of toxin A, NDGA, and toxin A plus NDGA on ileal LTB4 concentrations (N = 6). *P < .001 vs toxin A(−)/NDGA(−); #P < .001 vs toxin A(+)/NDGA(−). Gastroenterology 2005 128, 1306-1316DOI: (10.1053/j.gastro.2005.03.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Effects of NDGA on toxin A- and LTB4-stimulated ileal luminal fluid accumulation and MPO activity (N = 6). (A) Intraluminal pretreatment with NDGA 30 minutes before toxin A or LTB4 administration significantly inhibited toxin A-induced but not LTB4-induced luminal fluid accumulation. (B) Similarly, intraluminal pretreatment with NDGA 30 minutes before toxin A or LTB4 administration significantly inhibited toxin A-induced but not LTB4-induced MPO activity. *P < .01 vs NDGA(−)/toxin A(−)/LTB4(−); **P < .001 vs NDGA(−)/toxin A(−)/LTB4(−); #P < .001 vs NDGA(−)/toxin A(+)/LTB4(−). Gastroenterology 2005 128, 1306-1316DOI: (10.1053/j.gastro.2005.03.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 H&E-stained sections of rat ileal segments after they were treated intraluminally for 3 hours with (A) vehicle, (B) 1 μg of NDGA, (C) 5 μg of toxin A, (D) 5 μg of toxin A after pretreatment with 1 μg of NDGA 30 minutes before toxin A administration, (E) 3 μg of LTB4, or (F) 3 μg of LTB4 after pretreatment with 1 μg of NDGA 30 minutes before LTB4 administration. Scale bar, 100 μm. Gastroenterology 2005 128, 1306-1316DOI: (10.1053/j.gastro.2005.03.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 10 Proposed mechanism of toxin A-induced intestinal inflammation in the rat. A subtype of primary sensory neurons, with cell bodies in the dorsal root ganglia, express the TRPV1 receptor, which can be stimulated directly by capsaicin and resiniferatoxin (rtx) and indirectly by toxin A via generation of LTB4. TRPV1 stimulation results in SP release orthodromically in the spinal cord in laminae I and II to contribute to the transmission of pain signals and antidromically in the intestine to cause inflammation. Capsazepine and I-RTX are TRPV1 antagonists that directly block TRPV1 activation by capsaicin, RTX, and LTB4, and indirectly block the LTB4-mediated effects of toxin A. Gastroenterology 2005 128, 1306-1316DOI: (10.1053/j.gastro.2005.03.017) Copyright © 2005 American Gastroenterological Association Terms and Conditions