GPI-anchoring of GM-CSF results in active membrane-bound and partially shed cytokine.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce the generation and activation of dendritic cells (DCs), the most potent of antigen presenting cells (APCs). Tumors secreting GM-CSF have been shown to induce strong anti-tumor immune responses. In this report, we have constructed a glycosylphosphatidyl-inositol (GPI) anchored form of GM-CSF (GPI-GM-CSF). This protein subsequently was found expressed on the cell membrane and sensitive to phosphatidyl-inositol-specific phospholipase C (PIPLC), confirming that it is GPI-anchored. However, GM-CSF was also found in the culture supernatant of cells expressing GPI-GM-CSF. Inhibition studies using brefeldin A and para-formaldehyde fixation revealed that GM-CSF found in the supernatant was not secreted, but due to shedding or proteolytic cleavage. Accumulation of GM-CSF in the media from isolated membranes was time and temperature-dependent. The released portion represented 10-15% of all membrane-bound GM-CSF after 72h under culture conditions. GPI-GM-CSF retained functional activity to induce bone marrow cell proliferation and administration of GPI-GM-CSF expressing membranes induced the generation of DCs in vivo. These results demonstrate that GPI-anchored GM-CSF retains all functional activity of native GM-CSF while gaining the ability to attach to cell membranes. The ability of GPI-GM-CSF to be expressed on membranes and be partially released, can possibly lead to formation of a cytokine gradient, while retaining ability to target associated membrane antigens to DCs. This novel form of GM-CSF may have wide range of clinical applicability.
Poloso
Neil J
NJ
Department of Pathology and Laboratory Medicine, School of Medicine, Emory University, 1639 Pierce Drive, Woodruff Memorial Building, Room 7309, Atlanta, GA 30322, USA.
Nagarajan
Shanmugam
S
Mejia-Oneta
Jose M
JM
Selvaraj
Periasamy
P
D000818:Animals; D001854:Bone Marrow Cells / Q000502:physiology; D016466:CHO Cells; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003713:Dendritic Cells / Q000502:physiology; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D014162:Transfection
D017261:Glycosylphosphatidylinositols; D016178:Granulocyte-Macrophage Colony-Stimulating Factor