Activation of nuclear factor kappaB in colonic mucosa from patients with collagenous and ulcerative colitis

Abstract

Background and aims: Expression of inducible nitric oxide synthase (iNOS) is greatly upregulated in the colonic mucosa of patients with collagenous and ulcerative colitis. As the transcription factor nuclear factor kappaB (NFkappaB) is a major inducer of iNOS gene expression, we compared activation and transcriptional activity of NFkappaB in colonic mucosal biopsies from these patients.

Patients: Eight patients with collagenous colitis, six with relapsing ulcerative colitis, and eight with uninflamed bowel were studied.

Methods: NFkappaB DNA binding activity was assessed by electrophoretic mobility shift assay and inhibitor of NFkappaB (IkappaB) kinase (IKK) activity by immunocomplex kinase assay. In vivo recruitment of NFkappaB to the iNOS promoter was determined by chromatin immunoprecipitation analysis and transcriptional activity by NFkappaB gene expression profiling arrays. Cells showing NFkappaB activation were identified by immunohistochemistry.

Results: In collagenous and ulcerative colitis, as opposed to uninflamed bowel, IKKbeta activity and strong NFkappaB DNA binding gave rise to activation of identical NFkappaB subunits and recruitment of transcriptionally active p65 to the iNOS promoter. In collagenous colitis, activated NFkappaB was observed only in epithelial cells while up to 10% of lamina propria macrophages showed activation in ulcerative colitis.

Conclusions: In collagenous and ulcerative colitis, colonic mucosal NFkappaB is activated and recruited to the iNOS promoter in vivo via an IKKbeta mediated pathway. As collagenous colitis is not associated with tissue injury, these data challenge the prevailing view that activation of NFkappaB per se mediates tissue injury. Our results suggest that downstream inflammatory reactions leading to tissue damage originate in lamina propria immune cells, as increased NFkappaB activity in collagenous colitis was localised solely in epithelial cells, but present also in macrophages in ulcerative colitis.

Figure 1

(A) Nuclear factor κB (NFκB) DNA binding activity in nuclear extracts of colonic mucosal biopsies from patients with uninflamed bowel, collagenous colitis, or ulcerative colitis. Nuclear extracts were incubated with a ³²P labelled κB oligonucleotide probe and subjected to electrophoretic mobility shift assay (EMSA). Lanes 1, 2: patients with uninflamed bowel; lanes 3–5: patients with collagenous colitis; lanes 6, 7: patients with mild and severe ulcerative colitis, respectively. A representative result of two experiments is shown. (B) Nuclear extracts of colonic mucosal biopsies from patients with active ulcerative colitis or collagenous colitis were incubated with antibodies prior to EMSA analysis. An asterisk (*) denotes supershifted bands. A representative result of two experiments is shown.

Figure 2

In vitro IκB kinase (IKK) activities in cytosolic extracts of colonic mucosal biopsies from patients with uninflamed bowel (C), active collagenous colitis (COC), or ulcerative colitis (UC). (A, B) IKKα and IKKβ activities determined by immune complex kinase assay (KA) using cytosolic extracts of colonic mucosal biopsies incubated with glutathione S-transferase (GST)-p100 (754–900) (A) or GST-IκBα (1–54) (B) substrate and radioactive ATP. The specificity of phosphorylation was determined by comparing reactions with wild-type (WT) or mutant (MUT) substrates, in which serine 866 and 870 (A) or serine 32 and 36 (B) were replaced by alanine. The reaction mixture was resolved on an acrylamide gel, transferred to a polyvinylidene difluoride membrane and subjected to phosphorimager analysis. The membrane was subsequently probed with antibody against IKKα or IKKβ as a loading control using western blot analysis (WB). A representative result of six assays is shown.

Figure 3

Chromatin immunoprecipitation assay of p65 binding to a nuclear factor κB (NFκB) regulatory region within the inducible nitric oxide synthase (iNOS) promoter in nuclear extracts of colonic mucosal biopsies from patients with uninflamed bowel (C; n = 2), collagenous colitis (COC, n = 2), or active ulcerative colitis (UC, n = 2). Soluble chromatin was prepared from formaldehyde cross linked sonicated biopsies. Specific antibody against p65 was used to precipitate protein bound DNA fragments which were subsequently amplified by polymerase chain reaction using primers flanking an NFκB regulatory region (−5979 to −5779; primer pair A) or a non-regulatory region (−2741 to −2534; primer pair B). Total input refers to amplification of 1% of the total amount of DNA prior to immunoprecipitation.

Figure 4

Expression of inducible nitric oxide synthase (iNOS) mRNA in biopsies from colonic mucosa of patients with uninflamed bowel, collagenous colitis, or active ulcerative colitis determined by a microarray system and quantified by densitometry. Data were normalised by dividing individual densitometric values with the mean value of all housekeeping genes provided with the array. In the scatterplot, the logarithmic y axis denotes normalised iNOS mRNA expression, with group medians displayed as horizontal lines. *p<0.05 by one way ANOVA and Tukey’s test compared with uninflamed bowel.

Figure 5

Double immunohistochemical detection of activated nuclear factor κB (NFκB) and cellular markers for macrophages (CD68) in paraffin embedded formalin fixed biopsies from patients with uninflamed bowel (A, B), collagenous colitis (C, D), or ulcerative colitis (E, F). Negative NFκB staining (A) or weak but distinct focal staining of epithelial cells (B; arrows; red staining) was seen in uninflamed bowel. In collagenous colitis, NFκB nuclear staining was predominantly seen in the epithelium (C, D; arrows) while CD68 positive macrophages (C, D; arrowheads; blue staining) and CD68 negative stromal cells were left unstained. In ulcerative colitis, epithelial cells (E, F; arrows), CD68 positive macrophages (E, F; arrowheads), and CD68 negative stromal cells (E; broken arrows) showed intense nuclear expression of NFκB. Magnification ×100 (A–E) and ×150 (F).