Increased production of luminol enhanced chemiluminescence by the inflamed colonic mucosa in patients with ulcerative colitis.

Reactive oxygen species have been implicated as mediators of inflammation in ulcerative colitis. Chemiluminescence is a reliable means of estimating reactive oxygen species in biological media. Increased reactive oxygen species values in the inflamed colonic mucosa in rats were seen by chemiluminescence. The aims of the study were to find out if chemiluminescence is raised in the colonic mucosa of patients with ulcerative colitis and correlates with disease activity, and to elucidate the sources of the chemiluminescence. It was found that reactive oxygen species, as measured by the chemiluminescence technique, are raised in inflamed colonic mucosa and correlates with symptom score, sigmoidoscopic score, disease activity, and activity of the neutrophil enzyme myeloperoxidase. Chemiluminescence was inhibited by a myeloperoxidase inhibitor (azide) and an H2O2 scavenger (catalase) but not by allopurinol, an inhibitor of the enzyme xanthine oxidase. Chemiluminescence was also inhibited by indomethacin, but this did not seem to be related to inhibition of cyclo-oxygenase. These findings suggest that a likely cellular source of reactive oxygen species in the inflamed colon of patients with ulcerative colitis is the neutrophil and that myeloperoxidase conversion of H2O2 to hypochlorous acid, contributes to the chemiluminescence signal and possibly, to the tissue injury. Neither cyclo-oxygenase nor lipoxygenase seem to play a part as sources for the chemiluminescence.     

Mucosal blood flow and generation of superoxide in rat experimental colitis induced by succinic acid

As we consider succinic acid to be an exacerbating factor in ulcerative colitis, we investigated its influence on rat colonic mucosa in terms of mucosal blood flow and superoxide generation. We measured mucosal blood flow by the hydrogen gas clearance method and superoxide generation by the chemiluminescence method, and observed histopathological findings to determine the effects of succinic acid. After the instillation of succinic acid of any concentration tested to the colon, mucosal blood flow decreased. Histopathologically, the higher the concentration of succinic acid, the greater was the erosion formation in the colonic mucosa, while significant polymorpho-nuclear cell infiltration and superoxide generation from colon tissue were observed with 0.01% succinic acid compared with higher or lower concentrations. Succinic acid, at fecal concentrations found in active stage ulcerative colitis, appears to be implicated in mucosal injury, mediated by a decrease in colonic mucosal blood flow and infiltration of superoxide-generating polymorpho-nuclear cells into the mucosa.     

Excessive production of reactive oxygen metabolites by inflamed colon: analysis by chemiluminescence probe.

Reactive oxygen metabolites (ROMs) are involved in inflammatory diseases and are postulated to contribute to tissue injury in colitis. To determine whether excessive ROMs are generated by inflamed colonic mucosa and to identify possible sources and type of ROMs, mucosal ROMs were estimated in rats and humans using a chemiluminescence probe. Colitis was induced in rats by intracolonic injection of acetic acid or intraperitoneal injection of mitomycin C. Intact, inflamed colon in rats produced more ultraweak chemiluminescence than normal colon. Inflamed mucosal scrapings from both rat models produced significantly more luminol-enhanced chemiluminescence. Addition of catalase, an H2O2 scavenger, or azide, a myeloperoxidase inhibitor, into the media significantly decreased chemiluminescence from inflamed mucosal scrapings. Indomethacin, an antioxidant cyclo-oxygenase inhibitor, also decreased chemiluminescence, but MK-866, a 5-lipoxygenase inhibitor, had no effect. Colonic biopsy specimens obtained during colonoscopy from patients with ulcerative colitis also produced more catalase-inhibitable chemiluminescence than normal colonic mucosa. These data indicate that excessive ROMs are produced by inflamed colonic mucosa in both humans and rats, which may contribute to tissue injury.     

Agents capable of eliminating reactive oxygen species

Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, hydroxyl radical, and hypochlorous acid have been implicated in the pathogenesis of inflammation and tissue injury in colitis. To determine whether or not anti-ROS agents can decrease the severity of colitis, we evaluated the effects of three known anti-ROS agents: catalase, WR-2721, and Cu(II)₂(3,5-DIPS)₄ on acetic acid-induced colonic inflammation in rats. Histologically, all three compounds significantly decreased the severity of colonic inflammation. The anti-ROS activity of these compounds was also tested using the luminol-enhanced chemiluminescence assay. Catalase, WR-2721, or Cu(II)₂(3,5-DIPS)₄ significantly inhibited luminol-enhanced chemiluminescence produced by inflamed colonic mucosa. These findings suggest that ROS, and in particular superoxide, hydrogen peroxide, and/or one of its secondarily derived species, may play an important role in acetic acid-induced colitis. Further studies are needed to determine the potential effectiveness of these compounds in human colitis.     

Chemiluminescence assay of mucosal reactive oxygen metabolites in inflammatory bowel disease.

Previous studies suggesting increased reactive oxygen metabolite (ROM) production in inflammatory bowel disease have been restricted to peripheral blood and isolated intestinal phagocytes. In the current study, chemiluminescence and the effect of various scavengers, enzymes, and enzyme inhibitors were used to show that ROMs account for the increased production of oxidants by colorectal mucosal biopsy specimens in inflammatory bowel disease. Luminol-amplified chemiluminescence was increased in active ulcerative colitis [macroscopic grade 1: 25 photons.mg-1.min.10(-3) (median), 8-47 (95% confidence intervals), n = 40; grade 2: 89, 65-156, n = 30; grade 3: 247, 133-562, n = 13] and Crohn's disease [mild: 9, 3-84, n = 6; severe: 105, 25-789 (range), n = 5] compared with normal-looking mucosa (ulcerative colitis: 0.8, 0.4-1.4, n = 22, P less than 0.01; Crohn's disease: 0.8, 0.1-2, n = 6, P less than 0.05) and controls (0.6, 0.04-1.4, n = 52, P less than 0.01). In ulcerative colitis, luminol chemiluminescence correlated with microscopic inflammation (Spearman's p = 0.74, P = 0.0001) and was decreased by sodium azide (-89%, P less than 0.05), taurine (-31%, P less than 0.05), catalase (-23%, P less than 0.05), and dimethyl sulfoxide (-29%, P less than 0.05). Superoxide dismutase and oxypurinol decreased lucigenin chemiluminescence in ulcerative colitis by -63% (P less than 0.05) and -27% (P less than 0.05), respectively. Luminol chemiluminescence correlated with lucigenin chemiluminescence (Spearman's rho = 0.72, P = 0.003). These results suggest that neutrophil-derived oxidants (superoxide, hydrogen peroxide, hydroxyl radical, and hypochlorite) are generated in colorectal mucosa in active inflammatory bowel disease and support the hypothesis that production of such metabolites by neutrophils is of major pathogenetic importance.     

Evaluating the antioxidant potential of new treatments for inflammatory bowel disease using a rat model of colitis.

BACKGROUND: Reactive oxygen species may mediate tissue injury in inflammatory bowel disease. Aminosalicylates have antioxidant activity and the antioxidants, superoxide dismutase and allopurinol, are of reported benefit in inflammatory bowel disease. AIM: To develop a convenient technique for testing the antioxidant potential of standard and novel therapeutic agents for use in inflammatory bowel disease. METHODS: Amplified chemiluminescence was used to measure reactive oxygen species production by colonic biopsy specimens from rats with acetic acid induced colitis and to assess the in vitro effect of conventional antioxidants, standard therapies and proposed novel therapies for inflammatory bowel disease. RESULTS: The model was validated by demonstrating that the profile of effects on chemiluminescence of acetic acid induced colitis biopsy specimens given by conventional antioxidants (sodium azide, catalase, copper-zinc superoxide dismutase, dimethyl sulphoxide, N-acetylcysteine and ascorbate) and standard therapies (5-aminosalicylate and hydrocortisone) resembled that previously reported using biopsy specimens from ulcerative colitis. Human recombinant manganese superoxide dismutase did not alter chemiluminescence. Two novel compounds, LY231617 (10 mM) and amflutizole (20 mM), reduced chemiluminescence by 98% (n = 5, p = 0.009) and 88% (n = 5, p = 0.03), respectively. CONCLUSIONS: The similarity of the chemiluminescence responses of colonic biopsy specimens from acetic acid induced colitis and ulcerative colitis to a range of conventional antioxidants and standard treatments suggests that this model is a useful method for testing the antioxidant potential of new therapies for inflammatory bowel disease. The antioxidant actions of dimethyl sulphoxide, ascorbate, and the novel compounds, amflutizole and LY231617 in this model suggest that these agents merit further assessment in the treatment of inflammatory bowel disease.     

A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.

Acute and chronic experimental ulcerative colitis models were produced in mice by providing them with drinking water containing synthetic dextran sulfate sodium. Mice that developed acute colitis showed signs of diarrhea, gross rectal bleeding, and weight loss within 6-10 days after ingesting 3%-10% dextran sulfate sodium. On postmortem examination, multiple erosions and inflammatory changes including crypt abscesses were found on the left side of the large intestine. Mice that developed chronic colitis showed signs of erosions, prominent regenerations of the colonic mucosa including dysplasia, shortening of the large intestine, and frequent formation of lymphoid follicles after 5 administration cycles, where each cycle was composed of 7 days' consumption of drinking water containing 5% dextran sulfate sodium followed by 10 days' consumption of distilled water. The population of intestinal microflora, Bacteroides distasonis and Clostridium spp., increased significantly in mice with acute and chronic ulcerative colitis. Further, morphological studies suggest that the administered dextran sulfate sodium was partially phagocytized by macrophages in the colonic mucosa.     

Cytoprotection against neutrophil derived hypochlorous acid: a potential mechanism for the therapeutic action of 5-aminosalicylic acid in ulcerative colitis.

The aim of the present study was to investigate the effects of 5-aminosalicyclic acid (5-ASA) on the cell injury mediated by activated neutrophils. We used a system constituted of neutrophils, triggered with phorbol myristate acetate, and 51Cr-labelled Daudi cells as targets. The results show that 5-ASA is capable of efficiently preventing neutrophil-mediated lysis. 5-ASA was up to 10-fold more effective than taurine, which acts as an hypochlorous acid scavenger. Moreover, 5-ASA was found to compete with taurine for the neutrophil derived hypochlorous acid. The results are consistent with the conclusion that 5-ASA is capable of limiting the neutrophil mediated cell damage by scavenging the generated hypochlorous acid. This may represent a potential mechanism for the therapeutic action of 5-ASA in ulcerative colitis.     

Actions of sulfasalazine and 5-aminosalicylic acid as reactive oxygen scavengers in the suppression of bile acid-induced increases in colonic epithelial cell loss and proliferative activity

Sulfasalazine suppresses mucosal injury in patients with ulcerative colitis, but the mechanism of its therapeutic action is uncertain. In the present study, we examined the mechanism of the protective action of sulfasalazine in a rat model in which colonic epithelial cell loss and subsequent increases in epithelial proliferative activity were induced by intracolonic instillation of sodium deoxycholate. Sulfasalazine or its therapeutically active metabolite 5-aminosalicylic acid suppressed the loss of deoxyribonucleic acid into the colonic lumen and the subsequent increases in mucosal ornithine decarboxylase activity and tritiated thymidine incorporation into deoxyribonucleic acid induced by sodium deoxycholate. Sulfasalazine and 5-aminosalicylic acid also blocked xanthine-xanthine oxidase-induced loss of deoxyribonucleic acid and the subsequent proliferative response. In vitro sodium deoxycholate increased reactive oxygen formation by colonic mucosal scrapings or isolated crypt epithelium. These actions of sodium deoxycholate on reactive oxygen formation were blocked by sulfasalazine or 5-aminosalicylic acid. Sulfapyridine, a therapeutically inactive metabolite of sulfasalazine, had no effect on sodium deoxycholate-induced increases in surface cell sloughing, ornithine decarboxylase, tritiated thymidine incorporation into deoxyribonucleic acid, chemiluminescence, or superoxide production. The ability of sulfasalazine and 5-aminosalicylic acid to scavenge reactive oxygen may play a role in their therapeutic effects of inflammatory bowel disease.     

Platelet-Activating Factor,a Critical Mediator in the Pathogenesis of Dextran Sulfate Sodium-Induced Colitis in Rats

PURPOSE: Disorder of mucosal immunity based on an imbalance between proinflammatory and anti-inflammatory cytokines is believed to be a major factor in the pathogenesis of ulcerative colitis. Platelet-activating factor potentially stimulates the production of proinflammatory cytokines and recruits inflammatory cells. The aim of this study was to determine whether and to what extent platelet-activating factor plays a role in the pathogenesis of ulcerative colitis. METHODS: Using dextran sulfate sodium-induced colitis in rats as a model of ulcerative colitis, we analyzed the composition of cellular infiltrates and the local tissue expression of messenger ribonucleic acid for cytokine-induced neutrophil chemoattractant and tumor necrosis factor-alpha. To directly assess the impact of platelet-activating factor on the development of colitis, we also determined the efficacy of a specific platelet-activating factor receptor antagonist for preventing dextran sulfate sodium-induced colitis. RESULTS: The activity of colitis was well correlated with the upregulation of cytokine-induced neutrophil chemoattractant and tumor necrosis factor-alpha messenger ribonucleic acid in local tissues and infiltration of cytokine-induced neutrophil chemoattractant-positive neutrophils and ED1-positive macrophages. The platelet-activating factor receptor antagonist effectively ameliorated colitis, along with causing a decrease in the tissue cytokine-induced neutrophil chemoattractant messenger ribonucleic acid level and a decline in neutrophil and macrophage infiltration. However, the antagonist did not alter tissue levels of tumor necrosis factor-alpha messenger ribonucleic acid. CONCLUSION: Platelet-activating factor plays a critical role in the pathogenesis of dextran sulfate sodium-induced colitis through recruitment of cytokine-induced neutrophil chemoattractant-positive neutrophils and macrophages and/or stimulation of cytokine-induced neutrophil chemoattractant release from activated neutrophils. The tissue level of tumor necrosis factor-alpha messenger ribonucleic acid does not closely reflect the activity of colitis.     

Phospholipase A2 activating protein and idiopathic inflammatory bowel disease.

BACKGROUND: Crohn's disease and ulcerative colitis are idiopathic inflammatory bowel diseases (IBD) involving synthesis of eicosanoids from arachidonic acid (AA), which is released from membrane phospholipids by phospholipase A2 (PLA2). A potentially important regulator of the production of these mediators is a protein activator of PLA2, referred to as PLA2 activating protein (PLAP). AIMS: The purpose of this investigation was to discover if PLAP values might be increased in the inflamed intestinal tissue of patients with IBD and in intestinal tissue of mice with colitis. PATIENTS: Biopsy specimens were taken from patients with ulcerative colitis and Crohn's disease undergoing diagnostic colonoscopy, and normal colonic mucosa was obtained from patients without IBD after surgical resection. METHODS: Immunocytochemistry with affinity purified antibodies to PLAP synthetic peptides was used to locate PLAP antigen in sections of intestinal biopsy specimens from IBD patients compared with that of normal intestinal tissue. Northern blot analysis with a murine [32P] labelled plap cDNA probe was performed on RNA extracted from the colons of mice fed dextran sulphate sodium (DSS) and cultured HT-29 cells exposed to lipopolysaccharide (LPS). RESULTS: PLAP antigen was localised predominantly within monocytes and granulocytes in intestinal tissue sections from IBD patients, and additional deposition of extracellular PLAP antigen was associated with blood vessels and oedema fluid in the inflamed tissues. In contrast, tissue sections from normal human intestine were devoid of PLAP reactive antigen, except for some weak cytoplasmic reaction of luminal intestinal epithelial cells. Similarly, colonic tissue from DSS treated mice contained an increased amount of PLAP antigen compared with controls. The stroma of the lamina propria of the colonic mucosa from the DSS treated mice reacted intensely with antibodies to PLAP synthetic peptides, while no reaction was observed with control mouse colons. These data were supported by northern analysis which showed that PLAP mRNA was increased in the colons of DSS treated mice and cultured HT-29 cells exposed to LPS. CONCLUSIONS: As PLAP values were increased in the intestinal mucosa of IBD patients and mice with colitis, as well as in LPS treated cultured HT-29 cells, a role was postulated for PLAP in increasing PLA2 activity, which leads to the increased synthesis of eicosanoids in intestinal tissues of patients with these inflammatory diseases.     

Functional and phenotypical activation of leucocytes in inflamed human colonic mucosa

Infiltrating leucocytes are activated to generate reactive oxygen species or to produce several molecules in inflamed colonic mucosa. To clarify the phenotypical and functional properties of activating cells in colitic mucosa, 23 patients with ulcerative colitis and 13 controls were studied using a combined method for determining in situ nitroblue tetrazolium reducing activity and immunohisto-chemical characterization. Antibodies 25F9 (anti-macrophage), EG2 (anti-eosinophil cationic protein), MAC387 (anti-calprotectin, expressed by activated myeloid-histiocytes lineage), and MAC-1 (anti-CD11b) were used. The proportion of EG2, calprotectin, and CD11b-positive cells were significantly increased in inflamed mucosa. The proportion of EG2, calprotectin, and CD11b-positive cells significantly correlated with the histological degree of inflammation. Proportion of EG2-positive cells but not calprotectin nor CD11b-positive cells was significantly correlated with nitroblue tetrazolium reducing activity. Aggregated cells reducing nitroblue tetrazolium seen in severely inflamed mucosa were found to be EG2 positive. Most of the calprotectin-positive cells were 25F9 negative. In addition to activation of neutrophils and macrophages, eosinophil activation has been shown to be involved in inflamed colonic mucosa.     

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.     

ILEITIS AS A MAIN RECURRENT LESION IN A PATIENT WITH ULCERATIVE COLITIS: REPORT OF A CASE

We report a case of ulcerative colitis complicating ileitis that endoscopically and histologically resembled a colonic lesion. Eight years prior to the time of writing, the patient had undergone proctosigmoidectomy and ileocecal resection because of severe hemorrhagic lesions of ulcerative colitis. A month prior to the time of writing, bleeding from the stoma occurred. Endoscopy revealed erosions on easy‐bleeding mucosa in the ileum but no active inflammatory lesions in colonic mucosa except for small erosions in the descending colon beneath the stoma. Histologic findings of biopsy specimens from the ileal mucosa showed marked inflammation including neutrophile infiltration and crypt abscesses. This is a rare case of ulcerative colitis showing ileitis as a main recurrent lesion, suggesting that careful observation of the small intestine will be required after ileocecal resection in ulcerative colitis patients.     

Comparison of leukocyte excretion and blood loss in inflammatory disease of the bowel.

Clinical relapse of inflammatory bowel disease is characterised by increased neutrophil migration into the intestine. The site of the neutrophil chemoattractant(s), whether luminal or mucosal, may be important since, on contact with a chemoattractant, neutrophils cause indiscriminate damage to their immediate surroundings by generating reactive oxygen species and by lysosomal enzyme release. If this happens within the mucosa, inflammation should correlate significantly with tissue damage as assessed by bleeding, but if it occurs within the intestinal lumen, the inflammation would be disproportionately greater than the bleeding such as is seen in classical exudation. Intestinal inflammation and bleeding were quantitated with the simultaneous use of indium-111 labelled neutrophils (four day faecal excretion of indium-111) and chromium-51 labelled red cells in patients with ulcerative colitis (n = 12), Crohn''s disease (n = 15), and NSAID induced enteropathy (n = 34). Intestinal inflammation and blood loss correlated significantly (Spearman) in patients with ulcerative colitis (20.3% v 6.5 ml/d (median) r: 0.85, p < 0.001) and NSAiD enteropathy (1.6% v 1.9 ml/d, r: 0.60, p < 0.01) but not in Crohn''s disease (17.0% v 2.1 ml/d, r: 0.38, p > 0.1). For a given indium-111 excretion, patients with ulcerative colitis had significantly greater (p < 0.01) blood loss than patients with Crohn''s disease. These results suggest that the predominant site of neutrophil chemoattractants may be within the mucosa in ulcerative colitis and NSAID enteropathy and within the lumen in Crohn''s disease.     

Inhibition of neutrophil elastase prevents the development of murine dextran sulfate sodium-induced colitis

Background Neutrophil elastase (NE) is a major secretory product from activated neutrophils and a major contributor to tissue destruction. However, little is known about the pathogenic contribution of NE to ulcerative colitis (UC). This study was designed to investigate the contribution of NE by measuring NE activity in plasma and colonic mucosal tissue from UC patients and a murine acute colitis model, and to elucidate the therapeutic effect of the NE-specific inhibitor ONO-5046. Methods The NE enzyme activities in plasma and colonic mucosal tissue from UC patients were directly measured using an enzyme–substrate reaction. Acute colitis was induced in mice by administration of 1.5% dextran sulfate sodium (DSS) for 5 days. DSS-induced colitis mice were then treated with ONO-5046 (50 mg/kg body weight) intraperitoneally twice a day. Results In UC patients, the NE enzyme activity was significantly elevated in both the plasma and colonic mucosal tissue compared with healthy controls. In DSS-induced colitis mice, the NE enzyme activity increased in parallel with the disease development. ONO-5046 showed therapeutic effects in DSS-treated mice by significantly reducing weight loss and histological score. ONO-5046 suppressed the NE enzyme activities in both plasma and culture supernatant of colonic mucosa from DSS-induced colitis mice. Conclusions ONO-5046, a specific NE inhibitor, prevented the development of DSS-induced colitis in mice. NE therefore represents a promising target for the treatment of UC patients.     

Histochemical study of colonic cancer in experimental colitis of rats

A reliable test for premalignant lesions in the development of colonic cancer in chronic ulcerative colitis has been needed. Thus, we designed this cytochemical study, using a model of experimental colitis and colonic tumors induced in Wistar male rats by the feeding of dextran sulfate sodium. The colitis had histologic similarities to ulcerative colitis in man. The percent frequency of polypoid lesions (dysplasia or dysplasia with carcinomain situ) in the cecum and ascending colon was about 25% at three months and 90% at six months of dextran sulfate feeding. The cytochemical findings by high-iron diamine-Alcian blue staining andUlex europeus agglutinin binding were chronologically paralledled by histological changes in the colonic mucosa, and the binding pattern of peanut agglutinin was not different between normal and dextran-treated animals. Moreover, some cytochemical changes that occurred during the inflammatory responses were not present in dysplastic or malignant lesions. Thus, the histochemical tests were not useful for detecting of premalignant lesions earlier than by conventional histology. Nevertheless, the dextran sulfate model of colitis in the rat appears suitable for study of cancer development in ulcerative colitis.     

Effect of Sodium Butyrate on Reactive Oxygen Species Generation by Human Neutrophils

Background: Short-chain fatty acids enema has been shown to be effective in the treatment of ulcerative colitis (UC). However, the mechanisms that lead to this response have not been well characterized. The aims of this study were to investigate the effect sodium butyrate has on reactive oxygen species (ROS) generation by human neutrophils, which are responsible for mucosal injury. Methods: Human neutrophils incubated with or without sodium butyrate were stimulated with opsonized zymosan (OZ) or phorbol myristate acetate (PMA). ROS generation was largely differentiated with flow cytometry assays of hydroethidine oxidation and dichlorofluorescein oxidation for superoxide anion and hydrogen peroxide respectively, and luminol-dependent chemiluminescence for myeloperoxidase-mediated oxidants. Results:     

Protein kinase C activity of colonic mucosa in ulcerative colitis.

Protein kinase C (PKC) activity was measured in the inflamed colonic mucosa of 24 patients with ulcerative colitis and in the normal colonic mucosa of 10 patients with other benign diseases. The particulate fraction activity in ulcerative colitis mucosa was significantly increased compared with that of normal mucosa (320 +/- 47 versus 200 +/- 30 pmol/min/mg protein; p less than 0.05). Inflamed ulcerative colitis mucosa also showed significantly increased total PKC activity in the particulate fractions compared with normal mucosa (147 +/- 26 versus 37 +/- 8 pmol/min/mg tissue; p less than 0.05). Mucosal samples from ulcerative colitis patients were divided into 12 with mild and 12 with severe inflammation by histologic examination. The particulate PKC activity of severely inflamed mucosa was significantly lower than that of mildly inflamed mucosa (p less than 0.05). These results indicate that colonic inflammation in ulcerative colitis may be associated with altered cellular PKC activity.     

Oxidative mechanisms utilized by human neutrophils to destroy Escherichia coli

Serum-opsonized bacteria are efficiently ingested and killed by neutrophils within the phagocytic vacuole, where they are exposed to an array of reactive oxygen metabolites and toxic lysosomal components. Although bacteria may be destroyed by oxygen-independent mechanisms alone, many types of bacteria are not killed effectively unless they are attacked by oxygen metabolites. However, the apparent inability of extracellular scavengers, or inhibitors, of oxygen metabolites to gain access to the phagocytic vacuole makes this system difficult to evaluate. Therefore, we investigated the ability of neutrophils triggered with phorbol myristate acetate to destroy unopsonized E. coli in a serum-free model system. Neutrophils incubated with phorbol myristate acetate at a cell-to-bacteria ratio of 1:4 caused a greater than 95% reduction in colony-forming units (CFU) of E. coli in 60 min at 37 degrees C. Destruction of E. coli by the stimulated neutrophils was dependent on neutrophil number, stimuli concentration, and the incubation period. The neutrophil-mediated bactericidal effect was stimulated by superoxide dismutase, but was inhibited by catalase, azide, or compounds known to scavenge hypochlorous acid. Although stimulated neutrophils can generate long-lived endogenous N- chloroamines , these compounds did not play a direct role in destruction of E. coli in our model system. However, in the presence of exogenous iodide, endogenous N- chloroamines exerted a powerful bactericidal effect. Finally, neutrophils triggered with opsonized zymosan could also mediate E. coli destruction by a qualitatively similar process. Thus, we have demonstrated that neutrophils have the potential to utilize the myeloperoxidase system to generate bactericidal quantities of a species with characteristics similar to, if not identical with, hypochlorous acid.