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. 2002 Sep;51(3):379-85.
doi: 10.1136/gut.51.3.379.

Activation of signal transducer and activator of transcription (STAT) 1 in human chronic inflammatory bowel disease

S Schreiber  1 , P Rosenstiel, J Hampe, S Nikolaus, B Groessner, A Schottelius, T Kühbacher, J Hämling, U R Fölsch, D Seegert
Affiliations

Activation of signal transducer and activator of transcription (STAT) 1 in human chronic inflammatory bowel disease

S Schreiber et al. Gut. 2002 Sep.

Abstract

Background: Increased expression of proinflammatory cytokines, including tumour necrosis factor alpha, interleukin 6, and interferon gamma, as well as activation of proinflammatory signalling molecules such as nuclear factor kappa B, is characteristic of inflammatory bowel disease (IBD).

Aims: To investigate expression and activation of signal transducer and activator of transcription (STAT) 1 in patients with IBD.

Patients: Patients with active IBD (n=42), disease specificity controls (n=8), and normal controls (n=12) were investigated.

Methods: Expression and activation of STAT1 were assessed by western blotting and electrophoretic mobility shift assays in extracts of endoscopic colonic biopsies. Cellular localisation was determined by immunohistochemistry.

Results: Western blots and immunohistochemical staining revealed an increase in STAT1 expression and activation in mucosal samples from ulcerative colitis and to a lesser extend in Crohn's disease patients. High levels of suppressor of cytokine signalling (SOCS)-3 expression, an inhibitor of STAT activation, were observed in Crohn's disease patients and normal controls in western blot experiments whereas no differences were observed for SOCS-1 expression. Phosphorylated (p) STAT1 was mainly detected in monocytic cells and neutrophils in the inflamed mucosa. Induction of remission by systemic glucocorticoids led to a decrease in levels of pSTAT1. In vitro studies indicated a direct effect of steroid treatment on STAT1 activation.

Conclusions: Expression and activation of STAT1 are predominantly heightened in ulcerative colitis and may therefore play an important role in the pathophysiology of colonic inflammation.

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Figures

Figure 1
Figure 1
(A) Levels of signal transducer and activator of transcription 1 (STAT1) in the nuclear extracts of colonic biopsies. Extracts were examined by western blot with specific monoclonal antibodies. CD, Crohn's disease; UC, ulcerative colitis; IC, inflammatory controls. (B) Nuclear levels of STAT1 were determined by western blot in colonic mucosal biopsies from UC patients in relation to the histological degree of inflammation (grade 0, mild; grade 1, moderate; grade 2, severe). Representative of three experiments with identical findings.
Figure 2
Figure 2
(A) Phosphorylation of signal transducer and activator of transcription 1 (pSTAT1) in the intestinal mucosa from patients with ulcerative colitis (UC) or Crohn's disease (CD) and normal controls (N). STAT1 phosphorylation was expressed as the ratio of pSTAT1 to total STAT1. Every membrane was probed with an anti-PY701-STAT1 antibody and afterwards reprobed with a STAT1 specific antibody (an example is shown in (B)).
Figure 3
Figure 3
Representative electrophoretic mobility shift assay for assessment of signal transducer and activator of transcription (STAT)/DNA binding complexes in colonic mucosal biopsies from a control (N) subject, and two patients with ulcerative colitis (UC) or Crohn's disease (CD). The specificity of the complex was examined by cold competition with a 25-fold molar excess of unlabelled FcγRI-GAS (lane 4), β-casein-GAS (lane 5), nuclear factor kappa B (NF-κB) binding (lane 6), or activator protein 1 (AP-1) binding (lane 7) oligonucleotides. The existence of STAT proteins in the DNA binding complex was assessed by parallel use of different anti-STAT antibodies (lanes 8–12). The last lane represents a control with an isotype specific control antibody.
Figure 4
Figure 4
(A) Immunostaining for phosphorylated signal transducer and activator of transcription 1 (pSTAT1). Distinct staining with high numbers of positive cells were detected in ulcerative colitis (UC) in comparison with Crohn's disease (CD) or normal controls (N). The control shows a staining experiment with an irrelevant isotype antibody in a patient with ulcerative colitis. Arrowheads indicate the position of stained cells. (B) Double staining of UC biopsies with an anti-pSTAT1 antibody (green) and cell type specific antibodies (red) (open arrowheads, cells exclusively stained with the anti-pSTAT1 antibody; arrowheads: double stained cells; arrows, cells exclusively stained with cell type specific antibody).
Figure 5
Figure 5
Example of a suppressor of cytokine signalling 3 (SOCS-3) specific western blot in extracts from mucosal biopsies from normal control colon (N) or inflamed colonic regions from patients with Crohn's disease (CD) or ulcerative colitis (UC).
Figure 6
Figure 6
(A) Downregulation of signal transducer and activator of transcription 1 (STAT1) phosphorylation after therapy with glucocorticoids. Colonic mucosal STAT1 levels were examined before and 2–4 weeks after treatment of ulcerative colitis (UC) patients with systemic glucocorticoids (n=9) was initiated. Results were obtained by western blot and analysed densitometrically. (B) In vitro stimulation of isolated peripheral blood monocytes with interferon γ (IFN 1000 U/ml) and prednisolone (pred 10 μM) for the indicated times (30 minutes and two hours). pSTAT1 (top panel) was detected by western blot and total amounts of STAT1 were examined by reprobing of the same membrane (bottom panel).
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