FAS LIGAND IS PRESENT IN HUMAN ERYTHROID COLONY-FORMING CELLS AND INTERACTS WITH FAS INDUCED BY INTERFERON-GAMMA TO PRODUCE ERYTHROID CELL APOPTOSIS

Interferon gamma (IFN gamma) inhibits the growth and differentiation o f highly purified human erythroid colony-forming cells (ECFCs) and ind uces erythroblast apoptosis. These effects are dose- and time dependen t. Because the cell surface receptor known as Fas (APO-1; CD95) trigge rs programmed cell death after activation by its ligand and because in cubation of human ECFCs with IFN gamma produces apoptosis, we have inv estigated the expression and function of Fas and Fas ligand (FasL) in highly purified human ECFCs before and after incubation with IFN gamma in vitro. Only a small percentage of normal human ECFCs express Fas a nd this is present at a low level as detected by Northern blotting for the Fas mRNA and flow cytometric analysis of Fas protein using a spec ific mouse monoclonal antibody. The addition of IFN gamma markedly inc reased the percentage of cells expressing Fas on the surface of the EC FCs as well as the intensity of Fas expression. Fas mRNA was increased by 6 hours, whereas Fas antigen on the cell surface increased by 24 h ours, with a plateau at 72 hours. This increase correlated with the in hibitory effect of IFN gamma on ECFC proliferation. CH-11 anti-fas ant ibody, which mimics the action of the natural FasL, greatly enhanced I FN gamma-mediated suppression of cell growth and production of apoptos is, indicating that Fas is functional. Expression of FasL was also dem onstrated in normal ECFCs by reverse transcriptase-polymerase chain re action and flow cytometric analysis with specific monoclonal antibody. FasL was constitutively expressed among erythroid progenitors as they matured from day 5 to day 8 and IFN gamma treatment did not change th is expression. Apoptosis induced by IFN gamma was greatly reduced by t he NOK-2 antihuman FasL antibody and an engineered soluble FasL recept or, Fas-Fc, suggesting that Fas-FasL interactions among the ECFCs prod uce the erythroid inhibitory effects and apoptosis initiated by IFN ga mma. (C) 1998 by The American Society of Hematology.