Neutrophil derived human S100A12 (EN-RAGE) is strongly expressed during chronic active inflammatory bowel disease

Abstract

Background: Intestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC) is characterised by an influx of neutrophils into the intestinal mucosa. S100A12 is a calcium binding protein with proinflammatory properties. It is secreted by activated neutrophils and interacts with the multiligand receptor for advanced glycation end products (RAGE). Promising anti-inflammatory effects of blocking agents for RAGE have been reported in murine models of colitis.

Aims: To investigate expression and serum concentrations of S100A12 in inflammatory bowel disease (IBD).

Methods: We performed immunohistochemical studies and immunofluorescence microscopy in biopsies from patients with CD and UC. S100A12 serum concentrations were analysed using a sandwich ELISA.

Results: Immunohistochemical studies revealed profound expression of S100A12 in inflamed intestinal tissue from IBD patients whereas no expression was found in tissue from healthy controls. Staining for S100A12 during chronic active CD and UC was restricted to infiltrating neutrophils. Serum S100A12 levels were significantly elevated in patients with active CD (470 (125) ng/ml; p<0.001, n=30) as well as those with active UC (400 (120) ng/ml; p<0.01, n=15) compared with healthy controls (75 (15) ng/ml; n=30). Even in inactive disease, elevated serum concentrations were found, at least in CD. S100A12 levels were well correlated with disease activity in CD and UC.

Conclusions: We demonstrated that neutrophil derived S100A12 is strongly upregulated during chronic active IBD, suggesting an important role during the pathogenesis of IBD. Serum S100A12 may serve as a useful marker for disease activity in patients with IBD.

Figure 1

Expression of S100A12 in tissues from patients with active Crohn’s disease (CD) or ulcerative colitis (UC). Immunohistochemical staining showed extensive expression of S100A12 in inflamed colonic tissue of patients with active CD (A). S100A12 positive cells surrounded granulomatous lesions in CD (B). Similar local expression of S100A12 was found in UC (C). Numerous S100A12 positive cells assembled in crypt abscesses in UC (D). Staining of serial sections revealed colocalisation of S100A12 positive cells (E) and CD15 positive cells (F). In destructive crypt abscesses, S100A12 positive neutrophils transmigrated through the epithelium into the lumen (G). Immunofluorescence microscopy of double labelling studies with a-S100A12 Texas Red (red) and a-CD15-FITC (green) clearly proved expression of S100A12 by infiltrating CD15 positive granulocytes (H). Double labelled cells appear yellow due to summation of colours. The inserted figures in (H) show emission at a single wavelength for both fluorochromes. Scale bars indicate 100 μm.

Figure 2

Serum levels of S100A12 in bacterial infection, inflammatory bowel disease (IBD), and healthy controls. S100A12 levels were determined in 15 patients with severe bacterial infections, 40 patients with Crohn’s disease (CD), 34 patients with ulcerative colitis (UC), and 30 healthy controls. Symbols show individual serum levels; diamonds indicate mean values with 95% confidence intervals (error bars). *p<0.05, **p<0.01, mean differences from healthy controls. Patients with active IBD had S100A12 serum levels comparable with patients with severe bacterial infections/sepsis.

Figure 3

Individual follow up of S100A12 serum levels, erythrocyte sedimentation rate (ESR), and clinical disease activity. Individual courses of S100A12, ESR, and colitis activity index (CAI)/Crohn’s disease activity index (CDAI) in a patient with ulcerative colitis (UC) (A) and Crohn’s disease (CD) (B). Data are representative of 10 patients with inflammatory bowel disease.

Figure 4

Changes in S100A12 serum levels in Crohn’s disease (CD) patients after infliximab treatment. Individual courses of S100A12 and Crohn’s disease activity index (CDAI) in three patients before, two weeks, and four weeks after treatment with infliximab.

Figure 5

S100A12 in the supernatant of neutrophils after stimulation with tumour necrosis factor α (TNF-α). S100A12 was determined in supernatants of cells left untreated (w/o) or stimulated with TNF-α 2 or 5 ng/ml for 15 (15′) and 30 (30′) minutes, respectively. There was a time and dose dependent increase in S100A12 levels after treatment with TNF-α (**p<0.01; n=3).