Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin.
Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane. To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography. In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin. However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA). While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm. Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA). We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.
Rao
Mangala
M
Departments of Membrane Biochemistry and Blood Research, Walter Reed Army Institute of Research, Silver Spring, MD, USA. mangala.rao@na.amedd.army.mil
Peachman
Kristina K
KK
Alving
Carl R
CR
Rothwell
Stephen W
SW
D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D017951:Antigen Presentation / Q000187:drug effects; D000938:Antigen-Presenting Cells / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000032:analysis / Q000494:pharmacology*; D005260:Female; D005372:Filipin / Q000737:chemistry; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D008081:Liposomes; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010047:Ovalbumin / Q000378:metabolism* / Q000493:pharmacokinetics; D010587:Phagocytosis / Q000187:drug effects* / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects
D000924:Anticholesteremic Agents; D015395:Histocompatibility Antigens Class I; D008081:Liposomes; D005372:Filipin; D010047:Ovalbumin; D002784:Cholesterol