title: Interleukin-6 production in CD40-engaged fibrocytes in thyroid-associated ophthalmopathy: involvement of Akt and NF-κB.
abstract: OBJECTIVE
  CD40-CD40 ligand (CD40L) interactions appear to play pathogenic roles in autoimmune disease. Here we quantify CD40 expression on fibrocytes, circulating, and bone marrow-derived progenitor cells. The functional consequences of CD40 ligation are determined since these may promote tissue remodeling linked with thyroid-associated ophthalmopathy (TAO).
  
  
  METHODS
  CD40 levels on cultivated fibrocytes and orbital fibroblasts (GOFB) from patients with Graves' disease (GD), as well as fibrocyte abundance, were determined by flow cytometry. CD40 mRNA expression was evaluated by real-time PCR, whereas response to CD40 ligation was measured by Luminex and RT-PCR. Protein kinase B (Akt) and nuclear factor (NF)-kappa B (NF-κB) signaling were determined by Western blot and immunofluorescence.
  
  
  RESULTS
  Basal CD40 expression on fibrocytes is greater than that on GOFB. IFN-γ upregulates CD40 in both cell types and its actions are mediated at the pretranslational level. Fibrocytes produce high levels of cytokines, including interleukin-6 (IL-6), TNF-α, IL-8, MCP-1, and RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted) in response to CD40L. IL-6 induction results from an increase in steady state IL-6 mRNA, and is mediated through Akt and NF-κB activation. Circulating CD40(+)CD45(+)Col1(+) fibrocytes are far more frequent in vivo in donors with TAO compared with healthy subjects.
  
  
  CONCLUSIONS
  Particularly high levels of functional CD40 are displayed by fibrocytes. CD40L-provoked signaling results in the production of several cytokines. Among these, IL-6 expression is mediated through Akt and NF-κB pathways. The frequency of circulating CD40(+) fibrocytes is markedly increased in patients with TAO, suggesting that this receptor might represent a therapeutic target for TAO.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Kellogg Eye Center, Ann Arbor, Michigan 48105, USA.
authors.forename: Erin F
authors.initials: EF
authors.lastname: Gillespie
authors.forename: Nupur
authors.initials: N
authors.lastname: Raychaudhuri
authors.forename: Konstantinos I
authors.initials: KI
authors.lastname: Papageorgiou
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Atkins
authors.forename: Ying
authors.initials: Y
authors.lastname: Lu
authors.forename: Laya K
authors.initials: LK
authors.lastname: Charara
authors.forename: Tünde
authors.initials: T
authors.lastname: Mester
authors.forename: Terry J
authors.initials: TJ
authors.lastname: Smith
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Douglas
mesh_terms: D000328:Adult; D015153:Blotting, Western; D019013:CD40 Antigens / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D005500:Follow-Up Studies; D005786:Gene Expression Regulation; D049970:Graves Ophthalmopathy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D016328:NF-kappa B / Q000096:biosynthesis / Q000235:genetics*; D009915:Orbit / Q000473:pathology; D051057:Proto-Oncogene Proteins c-akt / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D019013:CD40 Antigens; D015850:Interleukin-6; D016328:NF-kappa B; D012333:RNA, Messenger; D051057:Proto-Oncogene Proteins c-akt