title: Abnormal tumor necrosis factor receptor I cell surface expression and NF-kappaB activation in tumor necrosis factor receptor-associated periodic syndrome.
abstract: OBJECTIVE
  Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autosomal-dominant autoinflammatory condition caused by mutations in the TNFRSF1A gene. The cellular mechanisms by which mutations in this gene trigger inflammation are currently unclear. Because NF-kappaB is the major intracellular signaling component inducing secretion of proinflammatory cytokines, we sought to determine whether differences in the clinical phenotype of patients with TRAPS may be attributable to variable effects of TNFRSF1A mutations on TNFRI expression, localization, or NF-kappaB activity.
  
  
  METHODS
  Peripheral blood mononuclear cells were obtained from patients (following informed consent), and cellular nuclear and cytosolic fractions were generated by subcellular fractionation. Localization of IkappaBalpha and NF-kappaB was determined by Western blotting of the resultant fractions. NF-kappaB subunit activity was determined by enzyme-linked immunosorbent assay analysis and confirmed by electrophoretic mobility shift assay. Subcellular localization of TNFRI was determined by immunofluorescence confocal microscopy or by immunoblotting following affinity isolation of plasma membrane by subcellular fractionation.
  
  
  RESULTS
  Cells from patients with the fully penetrant C73R mutation had marked activation of the proinflammatory p65 subunit of NF-kappaB. In contrast, cells from patients with the low-penetrant R92Q mutation displayed high levels of DNA binding by the p50 subunit, an interaction previously linked to repression of inflammation. Interestingly, although cells from patients with the C73R mutation have no TNFRI shedding defect, there was nonetheless an unusually high concentration of functional TNFRI at the plasma membrane.
  
  
  CONCLUSIONS
  High levels of TNFRI at the cell surface in patients with the C73R mutation hypersensitizes cells to stimulation by TNF, leading to increased NF-kappaB p65 subunit activation and an exaggerated proinflammatory response.
authors.affiliation: Barts and The London, Queen Mary's School of Medicine and Dentistry, University of London, London, UK.
authors.forename: Belinda
authors.initials: B
authors.lastname: Nedjai
authors.forename: Graham A
authors.initials: GA
authors.lastname: Hitman
authors.forename: Nasim
authors.initials: N
authors.lastname: Yousaf
authors.forename: Yuti
authors.initials: Y
authors.lastname: Chernajovsky
authors.forename: Susanna
authors.initials: S
authors.lastname: Stjernberg-Salmela
authors.forename: Tom
authors.initials: T
authors.lastname: Pettersson
authors.forename: Annamari
authors.initials: A
authors.lastname: Ranki
authors.forename: Philip N
authors.initials: PN
authors.lastname: Hawkins
authors.forename: Peter D
authors.initials: PD
authors.lastname: Arkwright
authors.forename: Michael F
authors.initials: MF
authors.lastname: McDermott
authors.forename: Mark D
authors.initials: MD
authors.lastname: Turner
mesh_terms: D000328:Adult; D002675:Child, Preschool; D010505:Familial Mediterranean Fever / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008297:Male; D052002:NF-kappa B p50 Subunit / Q000378:metabolism*; D019683:Penetrance; D010641:Phenotype; D017354:Point Mutation; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics* / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism*
chemical_list: D052002:NF-kappa B p50 Subunit; D047888:Receptors, Tumor Necrosis Factor, Type I; C506235:TNFRSF1A protein, human; D051996:Transcription Factor RelA