title: CD40 expression in normal human cornea and regulation of CD40 in cultured human corneal epithelial and stromal cells.
abstract: OBJECTIVE
  To determine whether CD40-CD40 ligand (CD40L) interaction plays a role in corneal inflammatory responses, the expression of CD40 and CD40L on normal human cornea was investigated. In addition, using cultured human corneal epithelial (HCE) and human corneal stromal (HCS) cells, the regulation of CD40 expression in human corneal cells investigated, including that induced by proinflammatory cytokines such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha.
  
  
  METHODS
  Frozen optimal cutting temperature (OCT) compound-embedded sections of corneal tissues obtained from 18 normal human corneas were examined by an immunoperoxidase staining technique with anti-CD40 and anti-CD40L monoclonal antibodies (mAbs). Also, cultured HCE and HCS cells, with IFN-gamma (250-1000 U/mL) or TNF-alpha (500-4000 U/mL) treatment for 1 to 4 days and with no treatment, were stained by the immunofluorescence technique with mAbs and analyzed by flow cytometry. RESULTS. The area of positive staining for CD40 showed a topographical difference. The limbal epithelial cells were predominantly positive for CD40. Positive staining was also found to a lesser extent on the cells in the basal layer of peripheral corneal epithelium. Epithelial cells of the central cornea showed no immunoreactivity for CD40. Corneal stromal cells were negative for CD40 in most of the donor tissues (positive: 5 of the 18 corneas). Endothelial cells were distinctly negative for CD40. Cultured HCE cells were also positive but decreased in positive cell number with lengthening culture period. None or less than 5% of the cultured HCS cells were CD40 positive. IFN-gamma enhanced CD40 expression on both cell types. In contrast, TNF-alpha enhanced CD40 on HCE but not on HCS cells. No component cells of normal human cornea or cultured HCE and HCS cells showed immunoreactivity for CD40L.
  
  
  CONCLUSIONS
  In the human cornea, CD40 is expressed predominantly on limbal epithelial cells and also on cultured HCE cells with high proliferative potential. In addition, the expression of CD40 is induced on cultured HCE and HCS cells differentially by proinflammatory cytokines, such as IFN-gamma and TNF-alpha.
authors.affiliation: Department of Ophthalmology, Nihon University School of Medicine, Tokyo, Japan. immuneiwata@hotmail.com
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Iwata
authors.forename: Koichi
authors.initials: K
authors.lastname: Soya
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Sawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Sakimoto
authors.forename: David G
authors.initials: DG
authors.lastname: Hwang
mesh_terms: D000911:Antibodies, Monoclonal; D019013:CD40 Antigens / Q000096:biosynthesis*; D023201:CD40 Ligand / Q000096:biosynthesis*; D002478:Cells, Cultured; D003315:Cornea / Q000378:metabolism; D003319:Corneal Stroma / Q000187:drug effects / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007124:Immunoenzyme Techniques; D007371:Interferon-gamma / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; D014409:Tumor Necrosis Factor-alpha; D023201:CD40 Ligand; D007371:Interferon-gamma