Abstract 585: Endotoxin Contamination of Apolipoprotein A1: Effect on Macrophage Proliferation---a Cautionary Tale

Abstract

Incubation of human macrophages with ApoA1 (purified from human plasma) for 1-2 days resulted in decreased cell density that was independent of cell toxicity. Although ApoA1 treatment caused macrophages to become more rounded and clustered, with some macrophages detaching, this did not account for the decrease in cell density. Thus, we considered that ApoA1 decreased macrophage cell proliferation. Using a BrdU incorporation cell proliferation assay, we demonstrated that ApoA1 (20 ug/ml) resulted in a >90% inhibition in macrophage proliferation. Using a recombinant version of ApoA1, we found that the results were dependent on the expression system: ApoA1 expressed in human cells did not inhibit proliferation but ApoA1 expressed in E. coli did. Human plasma-derived ApoA2 did not inhibit proliferation. All apolipoproteins were obtained commercially. We considered the possibility that endotoxin contamination of the apolipoproteins contributed to the differential inhibition of macrophage cell proliferation. Endotoxin alone very potently inhibited macrophage proliferation (0.1 ng/ml inhibited macrophage proliferation >90%). Measurement of endotoxin levels in the apolipoprotein products, including an analysis of free versus protein-bound endotoxin, suggested endotoxin mediated inhibition of macrophage proliferation. While human cell-derived recombinant ApoA1 and human plasma-derived ApoA1 showed similar levels of total endotoxin contamination (levels that were sufficient to inhibit macrophage proliferation), the levels of free endotoxin in the human cell-derived recombinant ApoA1 were more than 1000-fold less than levels in the human plasma-derived ApoA1. This finding helps explain why the recombinant ApoA1 did not inhibit macrophage proliferation, but the plasma-derived ApoA1 did. Conclusion: Our findings show that apparent apolipoprotein-mediated cell effects (or effects of any other biological products) can be significantly influenced by endotoxin contamination, especially when these products are tested on highly endotoxin-sensitive cells, such as macrophages. Lastly, endotoxin potently inhibits macrophage proliferation, which may be a significant pathogenic mechanism by which bacteria escape the immune system.