3-nitrotyrosine and Celiac-Disease

Inflammation is characterized by increased nitric oxide production. Nitrotyrosine has recently been suggested to be useful as a marker for NO-mediated tissue damage. In context of the development of an ELISA for detection of nitrotyrosine in plasma, monoclonal anti-nitrotyrosine antibodies were developed by injecting mice with nitrated keyhole limpet hemocyanin. The specificity of the antibodies was determined by binding to nitrated BSA, lack of binding to unmodified BSA, tyrosine, 3-chlorotyrosine or phenylalanine and inhibition of binding by nitrotyrosine. The antibodies developed are useful for Western blot analysis and immunohistochemical staining. Using these antibodies a nitrotyrosine sandwich ELISA was developed with a lower detection limit of approximately 0.2 nM. The intra- and interassay variance were 2.4% and 11.9%, respectively. Using this newly developed ELISA, 1.27 +/- 1.03 microM nitrotyrosine was detected in plasma samples of celiac disease patients whereas nitrotyrosine was undetectable in control samples. Elevated nitrotyrosine levels were paralleled by an increase in plasma concentrations of NO-oxidation products (NOx), nitrite and nitrate from 15.1 +/- 6.1 microM in controls to 61.0 +/- 28.2 microM in celiac disease patients. Both nitrotyrosine and NOx levels declined when the patients were on a gluten-free diet, suggesting a relation between intestinal inflammation and plasma nitrotyrosine and NOx levels.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Blood Proteins; Blotting, Western; Celiac Disease; Enzyme-Linked Immunosorbent Assay; Hemocyanins; Humans; Immunohistochemistry; Mice; Nitrates; Serum Albumin, Bovine; Tyrosine

Celiac disease is characterized by severe inflammation of the small intestine, and inflammation is known to often be associated with enhanced nitric oxide (NO) production. The aim of the present study was to determine whether children with active celiac disease show increased duodenal inducible nitric oxide synthase (iNOS) expression by immunohistochemistry. For this purpose, NO activity was assessed by detection of nitrotyrosine, which is an indicative marker for the formation of the NO- and superoxide-derived oxidant peroxynitrite. Serial staining with the macrophage markers CD68 and CD14 was performed to assess whether intestinal macrophages are involved in intestinal NO production. Duodenal biopsies from seven children with normal biopsy findings were used for comparison. Intense iNOS staining of enterocytes was observed in 10 of 11 celiac disease biopsies but in only 1 of 7 controls (p < 0.002). In addition, nitrotyrosine staining was detected in the enterocytes of celiac disease patients and was associated with iNOS staining. Moreover, the number of iNOS-positive cells in the lamina propria was significantly (p < 0.002) enhanced in patients with celiac disease. Serial staining for iNOS, CD68, and CD14 revealed an increase in CD68/CD14 double-positive monocytes and colocalization of iNOS and CD14 expression associated with this disease. Collectively, these data suggest that in patients with active celiac disease, synthesis of iNOS is induced in the intestine in association with the formation of peroxynitrite and nitration of cellular proteins. Furthermore, the increase in intestinal CD14-positive macrophages suggests a role for these cells in the pathophysiology of the disease.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Biopsy; Celiac Disease; Child; Child, Preschool; Enzyme Induction; Female; Humans; Immunoenzyme Techniques; Infant; Intestine, Small; Lipopolysaccharide Receptors; Male; Nitric Oxide Synthase; Tyrosine