Purpose: The role of inflammatory cytokines in the pathogenesis of necrotizing enterocolitis (NEC) is still undefined. Elevated levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha have been measured in infants with NEC, while elevated levels of nitric oxide (NO) have been reported in newborn infants with clinical sepsis. However, the cellular source of the NO or cytokines is unknown. The authors hypothesized that local intestinal production of NO induced by cytokines may contribute to the pathogenesis of bowel necrosis in NEC by inducing apoptosis (programmed cell death) or necrosis of the enterocytes. We examined the levels of inflammatory cytokines and NO in the intestine of infants undergoing surgical resection for NEC, and the cellular localization of human inducible NO synthase (NOS-2) in the inflamed gut.
Methods: We compared 15 patients undergoing bowel resection for NEC, with six infants (of similar age) undergoing intestinal resection for ileal atresia or stricture, meconium peritonitis, intussusception, or cecal perforation (control). Diagnosis of NEC was confirmed histologically. Representative segments of the surgical specimen were examined for messenger RNA (mRNA) for NOS-2 by Northern blotting and in situ hybridization. Cytokine mRNA was measured by polymerase chain reaction (PCR) because mRNA could not be detected by Northern blotting. The site of NO production was determined by in situ hybridization and immunohistochemistry. Apoptosis was measured using in situ DNA strand break extension (TUNEL). Nitrotyrosine immunoreactivity was assessed to determine if NO mediates cellular injury via peroxynitrite formation.
Results: Messenger RNA for NOS-2 was detected in nearly all patients with NEC except for one infant who underwent proximal diverting jejunostomy alone, and who did not have histological evidence of NEC at that site. NOS-2 mRNA was detected less frequently in control patients. In situ hybridization and immunohistochemistry showed that the enterocytes were the predominant source of NOS-2 activity in the intestine of NEC patients. Extensive apoptosis was seen in enterocytes in the apical villi of infants with NEC, and correlated with nitrotyrosine staining. NOS-2 activity was markedly diminished at the time of stoma closure, but remained elevated in infants who died from progressive disease. PCR showed variable cytokine mRNA expression in the intestine. Transforming growth factor (TGF)-beta expression was nearly identical in NEC and control. However, interferon (IFN)-gamma was present in 9 of 10 NEC, but only in one of six control patients.
Conclusion: The data show that NO is produced in large quantity by enterocytes in the intestinal wall of infants with NEC and leads to apoptosis of enterocytes in apical villi through peroxynitrite formation.