Activation of nuclear factor kappa B inflammatory bowel disease - PubMed (original) (raw)

Activation of nuclear factor kappa B inflammatory bowel disease

S Schreiber et al. Gut. 1998 Apr.

Abstract

Background: Expression of pro-inflammatory cytokines is increased in the intestinal lamina propria of patients with inflammatory bowel disease (IBD). Nuclear factor kappa B (NF kappa B) controls transcription of inflammation genes. On activation, NF kappa B is rapidly released from its cytoplasmic inhibitor (I kappa B), transmigrates into the nucleus, and binds to DNA response elements in gene promoter regions.

Aims: To investigate whether increased activation of NF kappa B is important in IBD and may be down-regulated by anti-inflammatory treatment.

Methods: Activation of NF kappa B was determined by western blot assessment and electrophoretic mobility shift assay in nuclear extracts of colonic biopsy samples as well as lamina propria mononuclear cells.

Results: Nuclear levels of NF kappa B p65 are increased in lamina propria biopsy specimens from patients with Crohn's disease in comparison with patients with ulcerative colitis and controls. Increased activation of NF kappa B was detected in lamina propria mononuclear cells from patients with active IBD. Corticosteroids strongly inhibit intestinal NF kappa B activation in IBD in vivo and in vitro by stabilising the cytosolic inhibitor I kappa B alpha against activation induced degradation.

Conclusions: In both IBDs, but particularly Crohn's disease, increased activation of NF kappa B may be involved in the regulation of the inflammatory response. Inhibition of NF kappa B activation may represent a mechanism by which steroids exert an anti-inflammatory effect in IBD.

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Figures

Figure 1

Figure 1

NF_κ_B p65 in nuclear extracts from colonic biospsy samples. Colonic biopsy specimens from 10 patients with active Crohn's disease (CD), 14 patients with active ulcerative colitis (UC), six disease specificity controls (DSC), and seven normal controls (NC) were extracted using a procedure to isolate the nuclear compartment. Densitometric readings from western blot assessments of NF_κ_B p65 are demonstrated by the bar graph (mean (SD)). Before electrophoresis, samples were adjusted to have equal contents of total protein. The inset shows a representative sample of the original blots. Individuals are marked on the blot with U (ulcerative colitis), C (Crohn's disease), D (disease specificity controls), or N (normal volunteer). The characteristics of the patients and localisation of the biopsy samples are shown in table 1_. The disease specificity control patients shown in the blot are patients 28,_ 25, 27, and 29 (from left to right).

Figure 2

Figure 2

Total NF_κ_B p65 and I_κ_B_α in colonic biospy tissue. Colonic biopsy specimens were homogenised and analysed by western blot without prior compartmentalised extraction. Densitometric readings are shown by the bar graph (mean (SD)). The filled bars show levels of total NF_κ_B p65 (left y axis), and the shaded bars levels of I_κ_B_α (right y axis). Before electrophoresis, samples were adjusted to have equal contents of total protein. No statistical differences were seen between patients with active Crohn's disease (CD, n = 6), patients with active ulcerative colitis (UC, n = 6), disease specificity controls (DSC, n = 6), and normal controls (NC, n = 5). The inset shows representative blots (C = Crohn's disease, U = ulcerative colitis, D = disease specificity control, and N = normal control). Biopsy specimens were taken from the same location as those used for nuclear extracts (fig 1).

Figure 3

Figure 3

Detection of NF_κ_B in the intestinal lamina propria. (A) Nuclear extracts were prepared from colonoscopic biopsy specimens. A representative radioactive electrophoretic mobility shift assay using consensus oligonucleotides to detect NF_κ_B in nuclear extracts from intestinal tissue is shown. In both Crohn's disease (n = 9) and ulcerative colitis (n = 8) increased levels of NF_κ_B were found in comparison with disease specificity controls (n = 5) and healthy volunteers (n = 8). (B) Nuclear extracts were prepared from LPMNCs, which were freshly isolated from colonic biopsy specimens. A blot from a representative non-radioactive gel shift experiment with ten samples (C = Crohn's disease; U = ulcerative colitis; D = disease specificity controls; N = normal controls) is shown. The highest levels of activated NF_κ_B were seen in patients with active Crohn's disease, with similar levels in all four samples. In only one of four normal control samples could low levels of oligonucleotide binding proteins be detected. Levels in patients with ulcerative colitis (four) as well as disease specificity controls (three) appeared to be increased as well, although not as much as in Crohn's disease. The presence of NF_κ_B p65 as part of the complex was controlled by supershift experiments (not shown). The levels of nuclear p65 determined in the same extracts by western blot are shown in the lower part of the figure.

Figure 4

Figure 4

Inhibition of NF_κ_B activation (A) and I_κ_B_α_degradation (B) by steroids (dexamethasone) and N-α_-tosyl-phenylalanine chloromethyl ketone (TPCK). Nuclear extracts were prepared from lamnia propria mononuclear cells obtained from colonoscopic biopsy samples (two patients with Crohn's disease and two normal volunteer controls). Cells were cultured for 30 minutes in the presence of lipopolysaccharide (LPS). A representative blot from a patient with Crohn's disease is shown. (A) Both dexamethasone (10 µ_M) and TPCK (30 µ_M) strongly reduced the amounts of NF_κ_B available for binding to consensus oligonucleotides. (B) I_κ_B_α _was assessed in total extracts by western blot. TPCK as well as dexamethasone stabilised I_κ_B_α against activation induced degradation.

Figure 5

Figure 5

Nuclear NF_κ_B p65 from colonoscopic biopsy specimens from patients with inflammatory bowel disease is decreased by steroid treatment. Sigmoid biopsy specimens were obtained from patients with moderately to highly active ileocolonic Crohn'_s disease and involvement of the sigmoid (CDAI 250_-450). Nuclear extracts were prepared and concentrations of NF_κ_B p65 were assessed by western blot. Seven days of treatment with steroids (60 mg prednisolone/day per os in addition to 5 mg betamethasone twice daily as an enema) induced a significant reduction (p = _0.0156) in nuclear NF_κ_B p65 concentration. I_κ_B_α levels remained unchanged (data not shown).

Figure 6

Figure 6

Schematic representation of NF_κ_B activation and inhibition by steroids. Activating stimuli including tumour necrosis factor α _(TNF-α) and lipopolysaccharide (LPS) promote degradation of I_κ_B_α _and the subsequent release of NF_κ_B p65 into the cytosolic compartment. NF_κ_B p65 translocates into the nucleus and forms dimers as part of the activation process. Functionally active NF_κ_B can then bind to specific sites in inflammation gene promoter regions and initiate transcription. Steroids appear to stabilise I_κ_B_α against activation induced degradation and thereby reduce the amount of functionally active NF_κ_B available in the nucleus.

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