Volume 115, Issue 2, Pages 357-369 (August 1998) Nuclear factor κB is activated in macrophages and epithelial cells of inflamed intestinal mucosa  Gerhard Rogler*, Korbinian Brand‡, Daniela Vogl*, Sharon Page‡, Robert Hofmeister*, Tilo Andus*, Ruth Knuechel§, Patrick A. Baeuerle∥, Jürgen Schölmerich*, Volker Gross*  Gastroenterology  Volume 115, Issue 2, Pages 357-369 (August 1998) DOI: 10.1016/S0016-5085(98)70202-1 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Pathway of NF-κB activation. In the cytoplasm, the NF-κB complex is associated with the inhibitory protein IκB. Activation of NF-κB is induced by a number of different signals as, for example, IL-1 and TNF-α. These signals activate an IKK complex that contains the IKKs IKK-α (IKK-1) and IKK-β (IKK-2). IKK-α and IKK-β are phosphorylated, leading to a recruitment of IκB and phosphorylation of IκB at serine32 and serine.36 This is followed by release of IκB from the complex. Consecutively, a rapid proteolytic degradation of IκB and a translocation of the activated NF-κB into the nucleus takes place. In the nucleus, the activated NF-κB dimer interacts with regulatory NF-κB elements in promoters and enhancers, leading to alterations in transcription rates and altered cell function. When the degradation of IκB is blocked by inhibitors of the proteasome complex, IκB reassociates with the NF-κB dimer, and NF-κB activation is inhibited. Gastroenterology 1998 115, 357-369DOI: (10.1016/S0016-5085(98)70202-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Immunofluorescence detection of activated NF-κB in the inflamed mucosa. Activated NF-κB (Cy3 fluorescence) was identified in the lamina propria or in crypts near the basal membrane in inflamed mucosa. In normal mucosa, only nonspecific autofluorescence was detected. (A) Normal mucosa. The crypts show autofluorescence (orange). No red fluorescence (activated NF-κB, Cy3) can be detected. (B) Diverticulitis. Specific red fluorescence is detectable mainly in the lamina propria (arrowheads), corresponding to nuclei of cells containing activated NF-κB. (C) Crohn's disease mucosa without macroscopic inflammation (score 0). Only autofluorescence can be seen. The autofluorescence in the lamina propria seems to be greater compared with (A) control specimens perhaps because of the deposition of collagen. (D) Crohn's disease with a low degree of inflammation (+). Activated NF-κB can be detected (arrowheads). (E) Ulcerative colitis without inflammation (0). Again mainly autofluorescence is visible. (F–H) Ulcerative colitis in a severe (+++) state of inflammation. A large number of stained nuclei is detectable (arrowheads). Positive cells are located in the lamina propria and in the crypts (original magnification: A–F, 400×; G, 250×; and H, 1000×). Gastroenterology 1998 115, 357-369DOI: (10.1016/S0016-5085(98)70202-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Immunohistochemical detection of activated NF-κB in the inflamed mucosa. Activated NF-κB was stained with the α-p65mAb and peroxidase DAB (brown reaction product). (A and B) Almost no cells containing activated NF-κB were detectable in control mucosa. (C and D) Activated cells were distributed in the lamina propria or close to crypts (arrows) in Crohn's disease with moderate inflammation (++). (E and F) The same was found in ulcerative colitis with severe inflammation (+++) (arrows). (G) Diverticultis with moderate inflammation (++). (H) A fibrotic stenosis from a surgical specimen of a patient with Crohn's disease was stained with eosin, and activated NF-κB was detected by the α-p65mAb and peroxidase BDHC (blue reaction product). Between the connective tissue depositions, nuclei with activated NF-κB can be detected (original magnification: A, B, E, and F, 400×; C, D, and G, 250×; H, 1000×). Gastroenterology 1998 115, 357-369DOI: (10.1016/S0016-5085(98)70202-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Activation score. Activation of NF-κB in uninflamed (0) and slightly (+), intermediate (++), or severe (+++) inflamed mucosal areas of control patients and patients with nonspecific colitis, diverticulitis, Crohn's disease, and ulcerative colitis. Data are expressed as means ± SEM. n.s./c, Not significant to control mucosa (0). *P < 0.001. **P < 0.01, significant to control mucosa. Gastroenterology 1998 115, 357-369DOI: (10.1016/S0016-5085(98)70202-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 NF-κB activation in uninflamed and inflamed areas of the same patients. Specimens were prestained only with eosin. Activated NF-κB was stained with the α-p65mAb and BDHC (blue granular reaction product). In uninflamed mucosa, the number of activated cells was clearly lower. An obviously greater number of activated cells (arrows) is present in the inflamed areas. (A) Nonspecific colonic inflammation (inflammation grading, 0). (B) Nonspecific colonic inflammation (inflammation grading, +). Activated NF-κB is clearly present in the basal nuclei of crypt epithelial cells (basal membrane marked). (C) Crohn's disease (inflammation grading, 0). (D) Crohn's disease (inflammation grading, +++). Activated NF-κB is found close to small blood vessels (red-stained erythrocytes). (E) Ulcerative colitis (inflammation grading, 0). (F) Ulcerative colitis (inflammation grading, +++). Activated NF-κB is again visible close to small blood vessels. Gastroenterology 1998 115, 357-369DOI: (10.1016/S0016-5085(98)70202-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Identification of the cell types containing activated NF-κB in the inflamed mucosa. The cellular marker antigens were visualized by the indicated antibodies and DAB (brown reaction product). Activated NF-κB was stained in a second step with the α-p65mAb and BDHC (granular blue reaction product). Activated NF-κB could be colocalized in macrophages with the (A–C) CD33 and with the (D) CD68 antibody and in (E) epithelial cells. (F) No clear activation could be detected in cells stained with CD3 (T cells). (A) Crohn's disease (inflammation grading, +++; CD33). (B) Ulcerative colitis (inflammation grading, +++; CD33). (C) Diverticulitis (inflammation grading, +++; CD33). (D) Ulcerative colitis (inflammation grading, +++; CD68). (E) Ulcerative colitis (inflammation grading, +++; EP4). (F) Ulcerative colitis (inflammation grading, +++; CD3). Gastroenterology 1998 115, 357-369DOI: (10.1016/S0016-5085(98)70202-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 EMSA for NF-κB and supershift assays. (A) EMSA. Nuclear extracts from lamina propria mononuclear cells from (a) normal and (b) inflamed mucosa (ulcerative colitis) and from epithelial cells from (c) normal and (d) inflamed mucosa (ulcerative colitis) were analyzed. Equal amounts of protein were loaded. A strong activation of NF-κB in (b) lamina propria mononuclear cells and (d) epithelial cells from inflamed mucosa could be detected. (B) Supershift analysis with exposure overnight. Nuclear extracts from lamina propria mononuclear cells were incubated with polyclonal antibodies against c-Rel (lane 1), p50 (lane 2), p65 (lane 3), or without antibody (lane 4) before EMSA. Incubation with anti-p65 (lane 3) almost completely supershifted the protein-DNA complexes. Incubation with anti-p50 (lane 2) and c-Rel (lane 1) weakened the NF-κB–DNA complex band. Again a clear difference between lamina propria mononuclear cells from normal and inflamed mucosa can be seen. (C) Supershift analysis with 4 hours of exposure. After 4 hours of exposure of nuclear extracts from lamina propria mononuclear cells from inflamed mucosa, the weakening of the NF-κB–DNA complex band by anti-p50 (lane 3) and anti-c-Rel (lane 4) is more visible. p65 (lane 2) almost shifts completely. Gastroenterology 1998 115, 357-369DOI: (10.1016/S0016-5085(98)70202-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 EMSA for NF-κB and supershift assays. (A) EMSA. Nuclear extracts from lamina propria mononuclear cells from (a) normal and (b) inflamed mucosa (ulcerative colitis) and from epithelial cells from (c) normal and (d) inflamed mucosa (ulcerative colitis) were analyzed. Equal amounts of protein were loaded. A strong activation of NF-κB in (b) lamina propria mononuclear cells and (d) epithelial cells from inflamed mucosa could be detected. (B) Supershift analysis with exposure overnight. Nuclear extracts from lamina propria mononuclear cells were incubated with polyclonal antibodies against c-Rel (lane 1), p50 (lane 2), p65 (lane 3), or without antibody (lane 4) before EMSA. Incubation with anti-p65 (lane 3) almost completely supershifted the protein-DNA complexes. Incubation with anti-p50 (lane 2) and c-Rel (lane 1) weakened the NF-κB–DNA complex band. Again a clear difference between lamina propria mononuclear cells from normal and inflamed mucosa can be seen. (C) Supershift analysis with 4 hours of exposure. After 4 hours of exposure of nuclear extracts from lamina propria mononuclear cells from inflamed mucosa, the weakening of the NF-κB–DNA complex band by anti-p50 (lane 3) and anti-c-Rel (lane 4) is more visible. p65 (lane 2) almost shifts completely. Gastroenterology 1998 115, 357-369DOI: (10.1016/S0016-5085(98)70202-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 EMSA for NF-κB and supershift assays. (A) EMSA. Nuclear extracts from lamina propria mononuclear cells from (a) normal and (b) inflamed mucosa (ulcerative colitis) and from epithelial cells from (c) normal and (d) inflamed mucosa (ulcerative colitis) were analyzed. Equal amounts of protein were loaded. A strong activation of NF-κB in (b) lamina propria mononuclear cells and (d) epithelial cells from inflamed mucosa could be detected. (B) Supershift analysis with exposure overnight. Nuclear extracts from lamina propria mononuclear cells were incubated with polyclonal antibodies against c-Rel (lane 1), p50 (lane 2), p65 (lane 3), or without antibody (lane 4) before EMSA. Incubation with anti-p65 (lane 3) almost completely supershifted the protein-DNA complexes. Incubation with anti-p50 (lane 2) and c-Rel (lane 1) weakened the NF-κB–DNA complex band. Again a clear difference between lamina propria mononuclear cells from normal and inflamed mucosa can be seen. (C) Supershift analysis with 4 hours of exposure. After 4 hours of exposure of nuclear extracts from lamina propria mononuclear cells from inflamed mucosa, the weakening of the NF-κB–DNA complex band by anti-p50 (lane 3) and anti-c-Rel (lane 4) is more visible. p65 (lane 2) almost shifts completely. Gastroenterology 1998 115, 357-369DOI: (10.1016/S0016-5085(98)70202-1) Copyright © 1998 American Gastroenterological Association Terms and Conditions