title: CD40 Expression in Fibrocytes Is Induced by TSH: Potential Synergistic Immune Activation.
abstract: BACKGROUND
  Fibrocytes appear to participate in inflammation and tissue remodeling in patients with thyroid-associated ophthalmopathy (TAO). These patients have increased frequencies of circulating TSH receptor (TSHR)- and CD40-positive fibrocytes, suggesting TSHR and CD40 may play roles in proinflammatory cytokine production, which ultimately leads to orbital inflammation and tissue remodeling.
  
  
  OBJECTIVE
  To investigate the potential interactions between the TSHR and CD40 signaling pathways and their roles in IL-6 and TNF-α production.
  
  
  METHODS
  CD40 expression on fibrocytes was assessed using flow cytometry; IL-6 and TNF-α protein release using Luminex technology; increased IL-6 and TNF-α mRNA abundance, using real-time PCR; TSH- and CD40 ligand (CD40L)-stimulated Akt phosphorylation in fibrocytes, by western blot analysis; TSHR-CD40 protein-protein interaction, using co-immunoprecipitation, and CD40-TSHR co-localization, using immunocytochemistry.
  
  
  RESULTS
  TSH enhances CD40 expression at a pre-translational level in fibrocytes. Production of IL-6 and TNF-α after costimulation with TSH and CD40L was greater than that after TSH or CD40L stimulation alone. TSH and CD40L costimulation also resulted in greater Akt phosphorylation. Akt and nuclear factor (NF)-κB inhibitors significantly reduced cytokine production after TSH and CD40L costimulation. TSHR and CD40L are colocalized on the cell surface and form a complex.
  
  
  CONCLUSIONS
  TSHR and CD40 in fibrocytes appear to be physically and functionally related. TSH stimulates CD40 production on the fibrocyte surface. Cytokine expression upon simultaneous stimulation of TSHR and CD40 is greater than levels achieved with TSH or CD40L alone. Increased expression of CD40 by TSH is a potential mechanism for this process.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan, 48105, United States of America.
authors.forename: Tünde
authors.initials: T
authors.lastname: Mester
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan, 48105, United States of America.
authors.forename: Nupur
authors.initials: N
authors.lastname: Raychaudhuri
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan, 48105, United States of America.
authors.forename: Erin F
authors.initials: EF
authors.lastname: Gillespie
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan, 48105, United States of America.|Department of Ophthalmology of Union Hospital, Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, People's Republic of China.
authors.forename: Hong
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan, 48105, United States of America.|Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, 48105, United States of America.
authors.forename: Terry J
authors.initials: TJ
authors.lastname: Smith
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan, 48105, United States of America.|Ann Arbor Veterans Administration Medical Center, Ann Arbor, Michigan, 48105, United States of America.
authors.forename: Raymond S
authors.initials: RS
authors.lastname: Douglas
mesh_terms: D019013:CD40 Antigens / Q000378:metabolism*; D016207:Cytokines / Q000096:biosynthesis; D005347:Fibroblasts / Q000276:immunology*; D006801:Humans; D011989:Receptors, Thyrotropin / Q000378:metabolism; D015398:Signal Transduction; D013972:Thyrotropin / Q000502:physiology*
chemical_list: D019013:CD40 Antigens; D016207:Cytokines; D011989:Receptors, Thyrotropin; D013972:Thyrotropin