title: Effects of endocytosis inhibitors on internalization of human IgG by Caco-2 human intestinal epithelial cells.
abstract: OBJECTIVE
  The purpose of this study was to characterize the internalization mechanism of human IgG into the epithelial cells of human small intestine, employing human intestinal epithelial cell line Caco-2 as an in vitro model system.
  
  
  METHODS
  Real-time PCR analysis and uptake studies of fluorescein isothiocyanate-labeled IgG (FITC-IgG) from human serum were performed using Caco-2 cells.
  
  
  RESULTS
  Real-time PCR analysis showed that mRNA level of the neonatal Fc receptor (FcRn) was increased during the differentiation process in Caco-2 cells. The binding of FITC-labeled human IgG to the membrane surface of Caco-2 cells increased with a decrease in pH of incubation buffer. The uptake of FITC-IgG was also stimulated at acidic pH and was time-dependent. The binding and uptake of FITC-IgG at pH 6.0 was partially, but significantly, decreased by human gamma-globulin in a concentration-dependent manner. A mixture of metabolic inhibitors (sodium azide and 2-deoxyglucose) significantly inhibited the uptake, but not the binding, of FITC-IgG. In addition, endosomal acidification inhibitors such as bafilomycin A(1) and chloroquine significantly increased the accumulation of FITC-IgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) and caveolin-dependent endocytosis inhibitors (nystatin and indomethacin) did not decrease the uptake of FITC-IgG at pH 6.0. In contrast, macropinocytosis inhibitors such as cytochalasin B and 5-(N-ethyl-N-isopropyl) amiloride significantly decreased the uptake of FITC-IgG at pH 6.0.
  
  
  CONCLUSIONS
  The internalization of human IgG in human intestine might be, at least in part, due to FcRn-mediated endocytosis, which could occur by a process other than clathrin- and caveolin-dependent mechanisms.
authors.affiliation: Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
authors.forename: Koya
authors.initials: K
authors.lastname: Sato
authors.forename: Junya
authors.initials: J
authors.lastname: Nagai
authors.forename: Naoko
authors.initials: N
authors.lastname: Mitsui
authors.lastname: Ryoko Yumoto
authors.forename: Mikihisa
authors.initials: M
authors.lastname: Takano
mesh_terms: D000963:Antimetabolites / Q000494:pharmacology*; D018938:Caco-2 Cells; D003847:Deoxyglucose / Q000494:pharmacology; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D011961:Receptors, Fc / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019810:Sodium Azide / Q000494:pharmacology; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D000963:Antimetabolites; D004791:Enzyme Inhibitors; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D012333:RNA, Messenger; D011961:Receptors, Fc; D019810:Sodium Azide; D003847:Deoxyglucose; C100688:Fc receptor, neonatal