Effects of pro-inflammatory cytokines on apolipoprotein E secretion by a human astrocytoma cell line (CCF-STTG1)

Apolipoprotein (apo) E has been implicated in Alzheimer's disease: however. little is known about the regulation of its secretion in astrocytes. To in vestigate the effects of pro-inflammatory cytokines such as interleukin-1 b eta (IL-I beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-ga mma (IFN-gamma) on apoE secretion by CCF-STTG1 cells, a sensitive and speci fic double sandwich Enzyme-Linked ImmunoSorbent Assay (ELISA) was developed . Using a monoclonal antihuman apoE antibody as the capture antibody, this assay was carried out with commercially available reagents, The assay had a sensitivity of 0.013 ng per well, within-run and between-run variation coe fficients of 6.0 and 8.6 per cent respectively. Then was no cross-reactions between antibodies used and apoAI, apoAII, apoB, apoCI, apoCII and apoCIII . Low apoE concentrations were assessed using a serum-free HepG2 culture me dium as secondary calibrator, containing 59 mu g l(-1) of apoE, In serum-fr ee medium, CCF-STTG1 cells secreted apoE, the accumulation of which in the cell medium increased linearly with time (27 mu g per 48 h). After 48 h of incubation, apoE secretion was inhibited by TNF-alpha but not affected by I L-1 beta and IFN-gamma. However, the effect of regulatory factors may depen d upon culture conditions since in the presence of 10 per cent fetal calf s erum, IFN-gamma significantly inhibited apoE secretion, Thus, apoE secretio n by CCF-STTG1 cells is inhibited by specific pro-inflammatory cytokines, T his new apoE ELISA presents the great advantage of using commercially avail able reagents which permit inter-laboratory comparability of results, invol ves relatively low cost and is adaptable for the measurement of low levels of apoE. Copyright (C) 2000 John Wiley & Sons. Ltd.