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THE ROLE OF GROUP IV PTK IN TNBS-COLITIS MODEL
Maitham Khajah, Donna-Marie McCafferty
Gastrointestinal Research Group, University of Calgary, Alberta
BACKGROUND: Fer and the closely related proto-oncogene Fps kinase are the only members of group IV family of non-receptor protein tyrosine kinase (PTK). We have previously shown in vivo that Fer kinase plays a key role in leukocyte recruitment and epithelial barrier dysfunction during endotoxin-induced inflammation.
AIM: In this study we examined the role of Fer and Fps kinase in a chemically-induced model of colitis. Also, we examined the role of group IV PTK in neutrophil functions (chemotaxis and superoxide production) in response to fMLP.
METHODS: Wild type (WT), Fer null (FerDR/DR), or Fps/Fer double deficient (FpsKR/FerDR) mice were used between 6-10 weeks of age. Colitis was induced by a single intrarectal injection of 0.1 ml TNBS (40 mg/ml in 20% ethanol). Healthy controls were also studied. Animals were killed at 3, 7, 10, or 14 days post-induction of colitis. Macroscopic and microscopic inflammatory scores were quantified and myeloperoxidase activity (MPO) as an indication of neutrophil recruitment was assessed. In vitro neutrophil chemotaxis toward bacterial-derived chemoattractant (fMLP) and leukotriene B4 (LTB4) was performed using the under-agarose chemotaxis assay. The kinetics of superoxide production in WT or Fer DR/DR neutrophils in response to fMLP was performed using cytochrome c reduction assay.
RESULTS: In WT mice, TNBS induced a significant increase in the macroscopic inflammatory score at 3 days (6.01±0.84) and 7 days (1.93±1.30) post induction of colitis relative to controls (0.36±0.01), but they had normal macroscopic score level at 10 days. A significant increase in macroscopic score was observed in FerDR/DR and FpsKr/FerDR relative to WT mice (p<0.05) at 3, 7, and 10 days. FerDR/DR mice had normal macroscopic score at 14 days, but the double deficient mice still had a significantly higher macroscopic score (p<0.05) at 14 days. The microscopic score and colonic MPO activity followed the same pattern seen in the macroscopic score of inflammation. A significant increase in Fer DR/DR (102.71±15.14) and FpsKr/FerDR (158.97±33.44) chemotaxis toward fMLP was observed compared to WT (48.87±8.29) neutrophils. The chemotactic response was the same in all groups toward LTB4. Superoxide production in WT neutrophils started 5 minutes after the addition of fMLP, but there was acceleration in the rate of superoxide production in FerDR/DR neutrophils starting immediately after the addition of the stimuli.
CONCLUSION: group IV PTK play a role in a chemically-induced model of colitis through modulating leukocyte recruitment and functions at the site of inflammation.