Increased interleukin-8 (IL-8) in rectal dialysate from patients with ulcerative colitis: evidence for a biological role for IL-8 in inflammation of the colon.

OBJECTIVE: Infiltration of neutrophils and their release of toxic reactive oxygen species (ROS) in the colonic mucosa are associated with tissue damage in ulcerative colitis (UC). This neutrophil migration may be induced by chemoattractants, such as cytokines, in the colonic milieu. One such chemoattractant is interleukin-8 (IL-8), a neutrophil chemokine that is present at high concentrations in inflamed mucosa. However, the functional significance of IL-8 in neutrophil attraction and activation in UC has not been established. We hypothesized that IL-8 in the colonic lumen of patients with UC primes neutrophils, leading to their attraction and activation. METHODS: The colonic milieu was sampled by rectal dialysis. Using a semi-permeable membrane with a molecular weight cut-off of 12 kDa, dialysis solution was placed in the rectum and allowed to equilibrate over a 4-h period with the colonic milieu of controls or of patients with UC. IL-8 concentrations were measured by ELISA. Two functions of healthy neutrophils (PMN) were measured: expression of CD11-b surface adhesion molecules (by flow cytometry), and production of ROS (by both chemiluminescence and cytochrome C reduction assays). Neutrophil functions after exposure to rectal dialysates or n-formyl-methionyl-leucyl-phenylalanine (fMLP) were assessed before and after adding anti-IL-8 antibody or the fMLP blocker BMLP. RESULTS: IL-8 concentrations in dialysates from patients with active UC were significantly higher than in controls and correlated with disease activity. UC dialysates significantly increased ROS production and CD11-b expression by neutrophils and anti-IL-8 antibody partially (50%) inhibited these stimulatory effects of UC dialysates. Preincubation of neutrophils with UC dialysates significantly potentiated the fMLP-induced rise in ROS and anti-IL-8 antibody completely abolished this priming effect. CONCLUSIONS: The colonic milieu, sampled by rectal dialysis, from patients with active UC can both activate and prime neutrophils in vitro. High concentrations of IL-8 in the colonic lumen of UC patients are partially responsible for the activating effects of rectal dialysates, and account for all of its priming effects. These findings provide direct evidence for a role for IL-8 in inflammatory bowel disease.

Modulatory effects of the colonic milieu on neutrophil oxidative burst: a possible pathogenic mechanism of ulcerative colitis.

An important hallmark of ulcerative colitis (UC) is mucosal neutrophil (PMN) infiltration associated with mucosal damage. This suggests that colonic chemoattractants such as bacterial products (e.g., N-formyl-methionyl-leucyl-phenylalanine (fMLP), lipopolysaccharide (LPS)) reach systemic circulation and attract PMNs to the colon. PMNs are then activated in the colonic mucosa and release their toxic oxidative metabolites. However, bacterial products are also present in the systemic circulation of healthy subjects. Thus we hypothesized that PMNs develop tolerance to colonic factors in the normal state and that this tolerance is absent in UC. We evaluated the PMN respiratory burst in response to stimulation with fMLP, LPS, or phorbol 12-myristate 13-acetate (PMA) by measuring the production of reactive oxygen species (ROS) with both luminol-enhanced chemiluminescence and a cytochrome C reduction assay. PMNs were obtained from control subjects, inactive UC patients, patients with UC who had undergone colectomies, and non-UC patients with colectomies. All three stimuli induced a significant rise in ROS. PMNs from non-UC colectomy subjects produced significantly higher ROS than PMNs from control subjects with intact colons in response to both fMLP and LPS. In contrast, PMNs from UC colectomy patients produced levels of ROS similar to those produced by PMNs from UC patients with intact colons in response to fMLP and LPS. Colectomy had no effect on PMA-induced ROS production in controls. The observed difference in fMLP-induced ROS production in control subjects with intact colons was not due to fMLP receptor down-regulation because a competition assay performed with the fMLP blocker BMLP showed a similar receptor apparent affinity in all four groups. We conclude the following: (1) the normal colonic milieu modulates the PMN respiratory burst, resulting in hyporesponsiveness of PMNs to "physiologic" but not "pharmacologic" stimulation. This effect is not due to receptor down-regulation. (2) UC colonic milieu does not appear to modulate PMN respiratory burst. This loss of PMN "tolerance" to colonic factors may have a pathogenic role in the sustained inflammation and tissue damage in UC.

Are dipyridamole (sensitive) calcium channels present in esophageal smooth muscle?

UNLABELLED: Calcium (Ca2+) entry from the extra-cellular space into the cytoplasm through voltage-dependent Ca2+ channels, specifically dipyridamole (DHP) sensitive ones (L-type), control a variety of biological processes, including excitation-contraction coupling in vascular and GI muscle cells. It has also been proposed that these channels may control esophageal contractility. However, DHP-sensitive Ca2+ channels in esophagus have not been well characterized biochemically. Thus, it is not known if these channels are similar in number or affinity to those in vascular or neural tissues--organs for which clinical use of calcium channel blockers has been successful. Thus, the purpose of this study was to identify and characterize DHP-sensitive calcium channels in esophagus and compare them to vascular, neural, and other GI tissues. METHODS: We carried out in vitro receptor binding assays on lower esophageal muscle homogenates, gastric and intestinal and colonic homogenates, and aortic muscle homogenates from ca; and on brain homogenates from rat. We used a radio-labeled dihydropyridine derivative [3H]nitrendipine, to label these sites and co-administration of unlabeled nimodipine to define specific binding. RESULTS: As expected, ligand binding to L-type Ca2+ channels in aortic vascular smooth muscle and brain was readily detectable: brain, Bmax=252 fmol/mg protein, Kd=0.88 nM; aorta, Bmax=326 fmol/mg protein, Kd=0.84 nM. For esophagus (Bmax=97; Kd=0.73) and for other GI tissues, using the same assay conditions, we detected a smaller signal, suggesting that L-type Ca2+ channels are present in lower quantities. CONCLUSION: L-type Ca2+ channel are present in esophagus and in other GI muscles, their affinity is similar, but their density is relatively sparse. These findings are consistent with the relatively limited success that has been experienced clinically in the use of calcium channel blockers for treatment of esophageal dysmotility.

The role of nitric oxide in ethanol-induced gastrointestinal dysfunction.

We recently showed that acute ethanol inhibits contractility of the lower esophageal sphincter (LES) and the lower esophageal body (LEB) both in vivo and in vitro. To evaluate the mechanism of this inhibitory effect of ethanol, we investigated the role of nitric oxide (NO) on contractility of isolated LES and LEB circular muscle strips using inhibitors of NO synthase (NOS), NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine. Ethanol significantly decreased LES basal tone. This effect was not mediated by NO, because inhibition was not prevented by inhibitors of NOS. Electrical field stimulation caused an On-response relaxation from LES strips, and an Off-response contraction from both LES and LEB strips. Inhibitors of NOS prevented the On-response relaxation of LES, but had no significant effect on LES Off-response contraction. Ethanol potentiated the On-response relaxation of the LES Off-response contraction. Ethanol potentiated the On-response relaxation of LES, but had no significant effect on Off-response contraction. Ethanol's potentiating effect of the On-response relaxation is NO-mediated, because it was abolished by NOS inhibitors. Ethanol also inhibited carbachol-induced LES contractility. This inhibitory affect was NO-mediated, because NOS inhibitors abolished it. Ethanol inhibited both the Off-response contraction and carbachol-induced contraction of LEB strips. These effects were not NO-mediated, because they were not affected by NOS inhibitor. These data suggest that NO is not a mediator for the inhibitory effect of ethanol on LEB contractility, and that NO seems to be a mediator of ethanol inhibition of some aspects of LES motor functions.

Ethanol inhibits contractility of esophageal smooth muscle strips.

Acute ethanol (EtOH) in vivo decreases both the pressure of the lower esophageal sphincter (LES) and the amplitude of contractions of the smooth muscle of the lower esophageal body (LEB) in both man and cat. However, the mechanism of this inhibitory effect of EtOH is unclear. This inhibitory effect could be caused by a direct effect of EtOH on the esophagus or be secondary to known inhibitory effects of EtOH on the central nervous system. To this end, we evaluated the in vitro effect of EtOH on contractility of smooth muscle strips from both LES and LEB. Circular muscle strips from LES and LEB were isolated from cats. Changes in resting tension of LES strips and changes in stimulant-induced tension of LES or LEB strips were measured in the presence of up to five concentrations of EtOH (12.5- 100 mM). Stimulants included electric field stimulation (EFS) and carbachol. EtOH at 75 mM significantly decreased resting LES tension. EtOH also decreased maximal contractile responses to carbachol in both LES and LEB and increased the EC50 of carbachol for LES, but not LEB. EtOH also modulated EFS-induced esophageal contractility; EtOH potentiated EFS-induced "on-response relaxation" in LES and decreased EFS-induced "off-response contractions" In LEB. EtOH-induced inhibition of esophageal contractility seemed to be reversible. EtOH did not result in muscle fatigue. Thus, EtOH can directly inhibit contractility of the esophagus, and does so reversibly and at pharmacologically relevant concentrations.