Regulation of inflammation by the Fps/Fes protein tyrosine kinase
Parsons, Sean Allan
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Fps/Fes and Fer are members of a distinct subfamily of cytoplasmic protein tyrosine kinases that have recently been implicated in the regulation of innate immunity. Previous studies showed that mice lacking Fps/Fes are hyper-sensitive to systemic lipopolysaccharide (LPS) challenge. This study identifies physiological, cellular and molecular defects that contribute to the hyper-inflammatory phenotype in Fps/Fes-null mice. We showed that plasma tumour necrosis factor (TNF) - levels were elevated in LPS challenged Fps/Fes-null mice as compared to wild type mice, and cultured Fps/Fes-null peritoneal macrophages treated with LPS showed increased TNF- production. Cultured Fps/Fes-null macrophages also displayed prolonged LPS-induced degradation of IB-, increased phosphorylation of the p65 subunit of NF-B, and defective TLR4 internalization. Next, we showed a role for Fps in the regulation of recruitment of inflammatory leukocytes. Using the cremaster muscle intravital microscopy model, we observed increased leukocyte adherence to venules, and increased rates and degrees of transendothelial migration in Fps/Fes-null mice, compared to wild type. There was also increased neutrophil migration into the peritoneal cavity subsequent to thioglycollate challenge. Using flow cytometry, we observed prolonged expression of the selectin ligand PSGL-1 on peripheral blood neutrophils from Fps/Fes-knockout mice stimulated ex-vivo with LPS. Finally, we examined the role of Fps/Fes in regulating apoptosis in response to inflammation. Upon intra-peritoneal challenge with LPS, Fps/Fes-null mice displayed a decreased depletion of macrophages from the peritoneal cavity. In response to ex-vivo TNF- stimulation, macrophages from Fps/Fes-null mice underwent decreased apoptosis and necrosis as assessed by flow cytometry. Immunoblot analysis revealed that Fps/Fes-null macrophages displayed more TNF--induced degradation of IB-α in Fps/Fes-null cells, with corresponding increases in the phosphorylation of the p65 subunit of NF-B. In addition, stimulation of macrophages with TNF-α up-regulated PARP expression in wild-type macrophages; this up-regulation was not observed in Fps/Fes-null macrophages. Finally we observed a decreased recruitment of macrophages to the peritoneal cavities of Fps/Fes-null mice, with a corresponding increase in neutrophil recruitment, 5 days after thioglycollate challenge. Overall, we show that there is a role for Fps/Fes in regulating inflammation at the physiological, cellular, and molecular levels, and that this might be relevant in inflammatory disease.