Gastroenterology

Gastroenterology

Volume 115, Issue 2, August 1998, Pages 357-369
Gastroenterology

Alimentary Tract
Nuclear factor κB is activated in macrophages and epithelial cells of inflamed intestinal mucosa

This article is dedicated to Professor Strohmeyer on occasion of his 70th birthday.
https://doi.org/10.1016/S0016-5085(98)70202-1Get rights and content

Abstract

Background & Aims: Transcription factors of the nuclear factor κB (NF-κB) family play an important role in the regulation of genes involved in inflammation. In inflammatory bowel diseases, proinflammatory cytokines known to be regulated by NF-κB are involved. The aim of this study was to investigate the role of NF-κB activation during mucosal inflammation in situ. Methods: A monoclonal antibody, α-p65mAb, was applied for immunofluorescence and immunohistochemical analysis that recognizes activated NF-κB. Electrophoretic mobility shift assay was used to directly demonstrate the presence of active DNA-binding NF-κB. Results: Using the α-p65mAb antibody, activated NF-κB could be found in biopsy specimens from inflamed mucosa but was almost absent in uninflamed mucosa. The number of cells showing NF-κB activation correlated with the degree of mucosal inflammation but was not significantly different between inflamed mucosa from patients with Crohn's disease, ulcerative colitis, and nonspecific colitis or diverticulitis. NF-κB activation was localized in macrophages and in epithelial cells as identified by double-labeling techniques. Electrophoretic mobility shift assay with isolated lamina propria mononuclear cells and epithelial cells confirmed these results. Conclusions: This study shows for the first time the activation of NF-κB during human mucosal inflammation in situ. In addition to macrophages, epithelial cells contained activated NF-κB, indicating an involvement in the inflammatory process.

GASTROENTEROLOGY 1998;115:357-369

Section snippets

Monoclonal antibody α-p65mAb

Production and characteristics of the monoclonal antibody α-p65mAb have been described in detail previously.30, 31, 32 The antibody was designed to bind to a region of the p65 subunit of NF-κB that is masked in the NF-κB–IκB complex. Only when IκB is released from the complex, binding of the α-p65mAb can occur. Therefore, it allows the exclusive identification of activated NF-κB (after IκB release) in situ. The antibody is suitable for immunohistochemical and immunofluorescence techniques

Patients

The cryostat sections used for the experiments were obtained from biopsy specimens of inflamed colon of 25 patients with ulcerative colitis, 23 patients with Crohn's disease, 4 patients with diverticulitis, 4 patients with nonspecific colitis, and 21 control patients. The control patients were older than the patients with inflammatory bowel disease (Table 1). None of the control patients was treated with corticosteroids, whereas 10 patients with Crohn's disease and 12 patients with ulcerative

Discussion

Recently Neurath et al. described an abrogation of experimental colitis in mice by local administration of p65 antisense oligonucleotides.11 They also examined the regulation of p65 in patients with Crohn's disease and found a significant up-regulation of p65 mRNA and protein levels in macrophages and endothelial cells from these patients compared with cells from normal mucosa. These studies were performed with isolated cells. Furthermore, the activation state of NF-κB was not addressed. In the

Acknowledgements

The authors thank E. Treher, W. Vogt, and S. Gruene for supplying biopsy samples.

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