Inflammatory bowel disease (IBD) comprises two diagnoses: ulcerative colitis (UC) and Crohn disease, both of which are chronic inflammatory diseases of unknown origin that afflict children and adults. In children, UC is usually diagnosed with symptoms of rectal bleeding and lower abdominal cramping on defecation. In contrast, signs of Crohn disease are often subtle, which may delay in diagnosis (1). When the diagnosis is established, the clinician depends on reliable markers of the ongoing inflammatory disease activity to individualize treatment. Direct inspection of the colonic mucosa using colonoscopy is routine but associated with disadvantages because the procedure is time-consuming, expensive, and may be painful. At Karolinska Hospital, general anesthesia is used to perform full colonoscopy in children. Currently, the pediatric gastroenterologist relies on reported symptoms and various blood chemistry tests that reflect systemic inflammatory response. This has obvious disadvantages because many children underreport their symptoms, and correlation between blood chemistry inflammation markers and local disease activity is poor (2,3). The need for better local markers of active inflammation in IBD is obvious.
Increased colonic nitric oxide (NO) generation has been demonstrated in UC and in Crohn disease (4–6). In inflammatory diseases, NO is produced by stimulation of the inducible isoform of NO synthase (iNOS) (7). In IBD, NO synthesis seems to occur in epithelial cells of the inflamed colonic mucosa, which has been shown to express iNOS (8). However, the role of NO in inflammation is not clear. In addition to its regulatory actions on vascular and intestinal smooth muscle, and on neuronal activity (9,10), NO exhibits active immunologic properties involved in host defense against microbes (11) and, at high concentrations, may be cytotoxic to mammalian cells (12,13).
We have previously demonstrated greatly increased luminal NO concentrations in patients with active IBD compared with healthy individuals (5). This indicates that luminal NO measurement is a feasible and useful method for detecting inflammatory disease process in the colon and a reliable method for monitoring inflammatory activity in IBD.
The aim of the present study was to evaluate whether rectal concentrations of luminal NO measured by chemiluminescence could be used to identify inflammatory disease activity of the colon in a pediatric population, after a diagnosis of IBD was established. Using an all-silicon balloon catheter for gas sampling, as recently described (13), we measured NO concentrations in the rectums of children with active or nonactive UC or Crohn disease of the colon, and in the rectums of a group of healthy children.
PATIENTS AND METHODS
Thirty-six children with IBD (mean age, 12.5 years; range, 4–17 years; 20 boys and 16 girls) who had had the disease for 1.6 years (range, 0–4 years) were included in the study. Fourteen of these had UC; one patient had proctitis, five patients had left-sided UC, and the remaining eight patients had extensive colitis (mean age, 12.6 years; range, 4–16 years). Twenty-two children had Crohn disease of the colon (mean age, 12.5; range, 4–17 years), and in six of these children, the terminal ileum was involved as well as the colon. Full colonoscopy verified the diagnosis, with histologic examination of mucosal biopsy specimens taken every 5 cm to 10 cm, resulting in 11 different concentrations. The Truelove–Witt criteria (14) was used to identify active disease (n = 6) in the UC population. The Harvey–Bradshaw (15) simple index was used to identify active disease (n = 12) among children with Crohn disease. The control group comprised 12 healthy children (mean age, 9.7 years; range, 4–16 years).
Determination of Rectal NO Concentrations
Nitric oxide was measured with a chemiluminescence analyzer (CLD 700; Eco Physics, Dürnten, Switzerland). The detection limit for NO was 1 part per billion (ppb). The analyzer was calibrated at known concentrations (100–10,000 ppb) of NO in nitrogen gas (AGA, Lidingö, Sweden), administered through an electromagnetic flow controller (Environics, Middletown, CT, U.S.A.). The chemiluminescence assay is highly specific for NO, with no interference from other nitrogen oxides (16).
All-silicone catheters were used for gas sampling. The catheter was inserted into the rectum, using lubrication gel free of local anesthetics, to a level 10 cm above the anal sphincter. The catheter balloon was inflated with 10 mL of ambient air, containing less than 5 ppb of NO, and left for 10 minutes to equilibrate with gases in the rectum. The equilibration period was selected using prestudy experiments (unpublished data). Then the gas was withdrawn from the catheter balloon and diluted to a final volume of 50 mL before chemiluminescence analysis with correction for dilution. Analysis was performed within 15 minutes.
Determination of Fecal NO Concentrations
Fresh fecal samples from four healthy children and four patients with IBD of the colon were incubated in a canister for 30 minutes. The all-silicone balloon catheter was inserted into the canister, and the balloon was filled with 10 mL of NO-free air and then permitted to equilibrate for 10 minutes.
Data are expressed as mean values ± standard error of mean (SEM) and range, where appropriate. Data were logarithmically transformed and compared using one-way analysis of variance followed by Bonferroni posttest. P less than 0.05 was considered significant.
The local research ethics committee at Karolinska Hospital approved the study. All patients and at least one of the parents gave informed consent.
Clinical Activity Index
Among the six children with active UC, three had mild disease and three had moderate disease, according to the Truelove–Witt criteria. Applying the Harvey–Bradshaw simple index, the mean score was 7.33 (range, 6–12) for the 12 children with active Crohn disease.
Rectal NO Concentrations
Healthy children had low concentrations of NO, 77 ± 17 ppb, in the rectum.
In children with nonactive UC, NO concentrations did not differ from those of healthy controls. In contrast, in patients with active UC, the NO concentrations increased approximately 100-fold compared with healthy controls (P < 0.001) and with those with nonactive UC (P < 0.05). The extent of colonic inflammation did not correlate with rectal NO concentrations (Fig. 1).
Children with nonactive Crohn disease of the colon displayed rectal NO concentrations similar to healthy controls and patients with nonactive UC. Children with active Crohn disease showed a great increase of luminal NO (P < 0.001), of similar magnitude to that found in children with active UC. Ileal involvement in addition to colonic disease did not affect the rectal NO concentration (Fig. 1).
In four children with Crohn disease, NO was analyzed during active disease and after a period of at least 4 months of nonactive disease. All showed lower NO concentrations when in remission (300 ± 143 ppb) than during exacerbation (17,830 ± 7,110 ppb) (Fig. 2).
No correlation was seen between C-reactive protein, erythrocyte sedimentation rate, or white blood cell count and rectal NO concentrations.
In fecal samples from healthy controls and patients with active IBD, only low concentrations of NO (136 ± 92 ppb) were found.
This study shows that NO concentrations in the rectum of children with active IBD are increased considerably when compared with concentrations in children with nonactive disease and those in healthy controls. The increased NO concentrations are readily detectable using gas sampling with a balloon catheter and chemiluminescence.
Nitric oxide plays a central role in various inflammatory responses in the body (17). In previous work, increased NOS activity, as measured indirectly by citrulline assay, was found in mucosal biopsy specimens from patients with active UC (4,18). Nitric oxide synthase activity was shown to be 10-fold higher in patients with UC and almost 4-fold higher in patients with Crohn disease compared with NOS activity in mucosal specimens from healthy controls (6). Chemiluminescence, as a direct method of measuring NO, has shown a more than 100-fold increase of luminal NO concentrations in UC and in Crohn disease (5). Therefore, data indicate increased NO generation in UC and Crohn disease. The greatly increased concentrations of NO recovered in the gut, but not in fecal samples, indicate enzymatic production of NO probably occurs through activation of iNOS, because the activity of this mammalian enzyme does not seem to be physiologically controlled after the enzyme is stimulated and expressed.
The cellular source of luminal NO in the gut is not known. Other groups, using a specific antibody directed against iNOS, have studied the distribution of the enzyme and singled out the inflamed epithelium as the site of NO synthesis in IBD (8,19). In addition to iNOS-positive epithelial cells around crypts, Mourelle et al. (20) found iNOS-positive granulocytes and macrophages in colonic tissue from patients with active UC. Ikeda et al. (21) later supported these findings, reporting iNOS-positive neutrophils and macrophages in mucosal biopsy specimens from patients with active UC. Immunohistochemical techniques clearly demonstrate iNOS positivity in inflamed intestinal tissue, whereas patients with IBD in remission display no immunoreactivity for iNOS (21). These findings support ours, showing increased NO concentrations in active IBD, but not in nonactive disease.
Systemic elaboration of NO in IBD has been determined by analyzing circulating concentrations of nitrite and nitrate (NOx), which are stable end-products of NO oxidation. In concordance with mucosal measurements of NO production, some studies show increased plasma concentrations in both active UC and Crohn disease (22,23). Other studies, however, have not verified these results (24). Therefore, local measurement of NO in the gastrointestinal tract seems to be a much more stable and sensitive marker of increased mucosal NO synthesis in IBD.
Whether NO exerts aggressive or protective actions in inflammatory reactions is debated (18,25,26). Possible proinflammatory effects of NO include stimulated chemotaxis of neutrophils and monocytes (27,28), and induced synthesis and release of proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1 (IL-1) (29,30). Tissue injury also may result from conversion of NO to peroxynitrite (31,32). Peroxynitrite exerts cytotoxic action through lipid peroxidation and sulfhydryl oxidation (33) and enhances a local inflammatory response (34). Other mechanisms by which NO can contribute to cell damage involve its inhibition of DNA synthesis through the targeting of ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis (35), and the generation of superoxide ions (36) through its role as a free radical. Nitric oxide also enhances vascular permeability, leading to local inflammatory edema (37). Nitric oxide elaboration, as induced by endotoxin, may also disturb the intestinal motility pattern in conjunction with an inflammatory response (38). Taken together, these mechanisms induced by increased NO production may be associated with the diarrhea seen in inflammatory reactions of the gut. As further evidence of the involvement of NO in IBD, glucocorticosteroids, the treatment of choice in this disease, are known to down-regulate iNOS expression (39). This glucocorticosteroid action may add to the antiinflammatory effect of these drugs. In a rat model of colitis, Rachmilewitz et al. (40) demonstrated amelioration of colitis by direct inhibition of the l-arginine / NO pathway using the l-arginine analogue NG-nitro-l-arginine methyl ester. Furthermore, in IBD with toxic megacolon, the inflammation has been associated with increased expression of iNOS in the colonic muscular layer (20).
In contrast with these potentially harmful effects of NO, increasing evidence indicates that NO could act as an endogenous inhibitor of immune responses. Nitric oxide has been shown to inhibit integrin-induced leukocyte adhesion (41). These data are consistent with results showing that NO donors are able to down-regulate leukocyte–endothelial cell adhesion (42). Furthermore, NO per se has been shown to down-regulate NF-κB, a transcription factor with key functions in the inflammatory process, which stimulates the expression of a variety of proinflammatory cytokines including IL-1, IL-2, IL-6, and tumor necrosis factor-α (43). In contrast with the study of Rachmilewitz et al. (40), Pfeiffer and Qiu (44), using the same type of experimental model for colitis in the rat, showed increased mucosal damage after administration of NG-nitro-l-arginine methyl ester, and ascribed protective properties to NO. In studies applying gene-knockout techniques, a more severe acetic acid–induced colitis developed in iNOS-deficient mice than in wild-type controls (45). Therefore, more knowledge about the role of NO in IBD must be gathered before pharmacologic intervention of the l-arginine / NO pathway can be considered for treating inflammatory processes.
So far, published data support a correlation between ongoing inflammatory activity and increased NO concentrations (5,13,46). Our current findings indicate that NO determination, feasibly performed as rectal measurements, is of value in differentiating between IBD in active and nonactive state. Because increased NO concentrations also occur in infectious colitis (47) and microscopic colitis (48), rectal NO measurements are unspecific in determining the cause of disease. However, we found no correlation between increased acute phase reactants and inflammatory activity of the gut. This is not surprising because such markers are regularly produced in the liver. Therefore, local measurement of NO in the gut is more specific in that it reflects the degree of inflammatory activity at a local site. When the diagnosis is established, rectal NO measurements may serve as a clinically feasible diagnostic tool in monitoring inflammatory disease activity in children with IBD.
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