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 investigate the effects of pro‐inflammatory cytokines such as interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) on apoE secretion by CCF‐STTG1 cells, a sensitive and specific double sandwich Enzyme‐Linked ImmunoSorbent Assay (ELISA) was developed. Using a monoclonal anti‐human 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 coefficients of 6·0 and 8·6 per cent respectively. There 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 medium as secondary calibrator, containing 59 μg l−1 of apoE. In serum‐free medium, CCF‐STTG1 cells secreted apoE, the accumulation of which in the cell medium increased linearly with time (27 μg per 48 h). After 48 h of incubation, apoE secretion was inhibited by TNF‐α but not affected by IL‐1β and IFN‐γ. However, the effect of regulatory factors may depend upon culture conditions since in the presence of 10 per cent fetal calf serum, IFN‐γ significantly inhibited apoE secretion. Thus, apoE secretion by CCF‐STTG1 cells is inhibited by specific pro‐inflammatory cytokines. This new apoE ELISA presents the great advantage of using commercially available reagents which permit inter‐laboratory comparability of results, involves relatively low cost and is adaptable for the measurement of low levels of apoE. Copyright © 2000 John Wiley & Sons, Ltd.