Effects of endocytosis inhibitors on internalization of human IgG by Caco-2 human intestinal epithelial cells.

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. Real-time PCR analysis and uptake studies of fluorescein isothiocyanate-labeled IgG (FITC-IgG) from human serum were performed using Caco-2 cells. 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. 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.