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

Abstract

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-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) 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 microg 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 microg per 48 h). After 48 h of incubation, apoE secretion was inhibited by TNF-alpha but not affected by IL-1beta and IFN-gamma. However, the effect of regulatory factors may depend upon culture conditions since in the presence of 10 per cent fetal calf serum, IFN-gamma 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.