In this study we performed a kinetic analysis of gut mucosal NO production in relation to the local inflammatory response induced by instillation of wheat gluten into the rectum of patients with CD. This was made possible by the use of a newly developed technique (mucosal patch technique) that allows simultaneous measurements of NO and granule proteins released from inflammatory cells into the gut lumen. The obvious advantage of this approach is the gentle handling of the mucosa and the reduced distress for the patient compared with a study with repeated rectal biopsies, which also in fact, might influence NO synthesis.14
Our main finding was that signs of granulocyte activation clearly preceded the increased synthesis of NO after gluten challenge. Increased NO synthesis was not apparent five hours after gluten instillation, a time point with maximum release of MPO, a granule constituent of neutrophils, and of ECP, a granule constituent of eosinophils. Luminal NO values peaked 15 hours after gluten challenge and then gradually declined, but were still increased after 48 hours. The granulocyte activation pattern described a biphasic pattern compatible with previous histological findings reporting a biphasic mucosal influx of neutrophils after rectal gluten challenge in coeliac patients.7
This pattern might possibly reflect the fact that the gluten induced reaction involves both the adaptive as well as the innate system.11
NO is generated via NOS which has constitutive and inducible isoforms. The constitutive isoform of NOS (cNOS) is calcium dependent and involved in various physiological conditions. In response to physiological stimulation, for example of endothelial cells and neurones, NO is generated rapidly and transiently at low concentrations for purposes such as relaxation of vascular smooth muscle cells leading to vasodilatation and inhibition of leucocyte and platelet adhesion to vascular endothelium. The major inducible isoform of NOS (NOS II) produces NO in high concentrations for as long as the enzyme is activated as part of immune and inflammatory responses. This isoform is constitutively present in the epithelium of some tissues but is mainly expressed by inflammatory cells (macrophages, eosinophils, mast cells, and possibly also neutrophils and T lymphocytes15–19
) on activation by proinflammatory cytokines and other inflammatory mediators. The role of NOS II in the inflammatory response to gluten instillation in the rectal mucosa has previously been elucidated by measurement of rectal mucosal biopsy samples obtained 4, 8, 24, and 48 hours after rectal gluten challenge.20
A significant (approximately 50%) increase in NOS II was seen eight hours after gluten challenge but there was no change in cNOS. Increased NO production in the gut lumen has been observed not only in ulcerative colitis21
but also 24 hours after gluten challenge in patients with CD.22
Our finding of high production of NO starting 5–10 hours after gluten challenge is in accordance with the above mentioned biopsy results concerning NOS II expression,20
as this expression has been found to precede the production of NO by several hours; the delay in NO production reflecting the time taken for mRNA and protein synthesis.23,24
NOS II expression in rectal biopsy specimens gradually decreased and was lost 24–48 hours after challenge. Our NO measurements showed a return to prechallenge NO values in 30% of our patients but elevated NO values still remained in a few patients even 48 hours after the challenge. In bronchial asthma an increase in luminal airway NO is also seen and it has been suggested that this may be useful for non-invasive determination of airway inflammation.25
After allergen challenge in asthmatic patients, no increase in NO was observed in those who responded only with an acute reaction, while those who also had a late allergic response had a peak increase in NO at 10 hours and still showed elevated NO levels 21 hours after the challenge. The similarity to our NO reaction strengthens the idea that gut and airway luminal NO production might be a non-specific inflammatory response.
The cellular source of induced luminal NO synthesis has not been identified in asthmatic patients. There is evidence of increased expression of NOS II in asthmatic airways, especially in macrophages and epithelial cells, but activated neutrophils, eosinophils, and mast cells have also been proposed as major contributors to enhanced NO synthesis.17,26,27
The early inflammatory reaction induced by gluten in patients with CD is characterised by expression of E-selectin, a specific adhesion molecule that is the main mediator involved in neutrophil recruitment in the first four hours after gluten exposure.28
This is in concordance with our observations that a strong neutrophil activation, as assessed by luminal release of MPO, and less pronounced eosinophil activation, as measured by release of ECP, was already present five hours after the gluten instillation challenge and clearly preceded the NO response.
Gluten induced small intestinal mucosal inflammation is known to be patchy. Our technique is designed to reduce the influence of an uneven distribution of inflammatory signals by allowing measurements at different mucosal sites. Nevertheless, the observed interindividual variability in NO and MPO responses may partly be due to non-homogenous inflammation. Another, perhaps more likely, explanation is individual sensitivity.29
The finding of a significant correlation between neutrophil and NO responses after challenge demonstrates that NO synthesis is linked to the intensity of the induced inflammatory reaction, but only suggests that neutrophils are the cellular NO source.
NO in exhaled air (eNO) has been proposed as a marker of bronchial inflammation. Some studies reported a significant association in patients with bronchial asthma between eNO and airway eosinophilia, as reflected by sputum eosinophilia30,31
or density of eosinophilic granule constituents in airway mucosa. Because eosinophils can express NOS II,32
these cell have been attributed a role in the synthesis of NO after allergen challenge. However, others have been unable to confirm a relationship between eNO and airway eosinophilia in asthma.33,34
In the present study, signs of eosinophil activation, as reflected by ECP, were present after gluten challenge in patients with CD, with a similar time course of activation as neutrophils, but the eosinophil response was relatively weaker than the neutrophil response defined by MPO. The lack of relationship between the degree of eosinophil activation and NO production may suggest that NO reflects other aspects of gut mucosal inflammation, including neutrophil and possibly also T cell and monocyte activation.
The site of NOS II expression in the epithelium of gluten damaged rectal mucosa is controversial but immunohistochemical studies of such mucosa showed that NOS II was mostly localised in the lamina propria just beneath the surface epithelium and around the crypts.20
The suggested protective effect of NO in inflammation has been partly attributed to a role in reducing granulocyte infiltration and consuming oxygen species.3
Thus the observed relationship between signs of neutrophil activation and later NO synthesis may well reflect a counteracting system controlling the potentially tissue damaging principles delivered by activated neutrophils. However, reports suggesting a protective role of NO in mucosal inflammatory damage are as numerous as those supporting a toxic role. It has been proposed, for example, that NO may promote inflammation by enhancing vascular permeability and by promoting chemotaxis of granulocytes and the production of proinflammatory cytokines.35,36
Others have suggested that epithelial cells are the cellular source of luminal NO seen in bronchial asthma and inflammatory bowel diseases.37,38
Our kinetic study is compatible with this hypothesis as activated granulocytes may induce enterocytes to increase NO synthesis. The results of our study give no answers concerning the possible toxic or protective role of NO in gluten induced damage. The possibility also remains that the association between granulocyte activation and NO production merely reflects the metabolic expression of neutrophils/eosinophils and other inflammatory cells activated by gluten challenge.
The lack of luminal histamine release after gluten challenge indicates that mast cells/basophils are not involved in induced NO synthesis. Niveloni et al
observed in biopsy samples that the number of NOS II positive cells after gluten exposure increased in parallel with the CD3 cell infiltration observed around the subepithelial areas.20
T cells have been attributed a central role in the pathogenesis of gluten induced mucosal damage but the NO producing capacity of T cells in inflammation remains unsolved.
The observation that corn gluten challenge induced an abnormal NO reaction in some of our patients with CD is intriguing as maize is considered safe and is recommended as the substitute cereal in a gluten free diet. However, a high incidence of serum antibodies against maize39
has been reported in CD, and failure to normalise the mucosa in a fraction of adult patients with CD and on a strict wheat gluten free diet remains to be explained.40
The manufacturer claimed that their corn product was free from wheat or other cereals. We tested the product at the Swedish National Food Administration (Livsmedelsverket) and it was found to be contaminated with 82 μg/g (ppm), which is less than the usual allowed amount in a gluten free diet (<200 ppm) according to the Codex Alimentarius Standard for gluten free foods, and far less than what has been found to be a safe amount of gluten contamination when correlated with histology in oral challenge studies.41
It cannot be excluded that the small amounts of gluten present in the corn preparation induced an inflammatory reaction as the mucosal patch technique is very sensitive.
The major finding in the present study was that mucosal activation of neutrophils and eosinophils precedes pronounced enhancement of mucosal NO production after rectal wheat gluten challenge in CD. The observations were made possible by the use of a simple and safe procedure, which may also be of value for elucidating the possible role of other food antigens, such as milk and egg proteins, that may cause symptoms and mucosal damage in some patients with CD.