Albumin does not induce IL-6 release and toll-like receptor activation in vitro: Role of endotoxin contamination and biochemical modifications.

Human serum albumin (HSA) is widely used in both clinical and research settings, yet its potential to stimulate immune responses remains poorly understood. In this study, we systematically investigated various therapeutic, recombinant and plasma-derived albumin preparations for their ability to induce pro-inflammatory cytokine release by human peripheral blood mononuclear cells (PBMC) and activate Toll-like receptors (TLR) in HEK-Blue™ TLR4 and TLR2 reporter cell lines. All albumin preparations were characterized in terms of their redox state of cysteine-34, glycation level, non-esterified fatty acid content, and endotoxin contamination Therapeutic albumin, as may be expected, did not induce IL-6-release nor TLR activation. However, several commercial research grade albumins, including fatty acid-free, glycated, and recombinant preparations, significantly induced IL-6 release and activated TLR4 and TLR2 signaling. These inflammatory effects were strongly correlated with the endotoxin content, while no consistent associations were found with albumin redox state, degree of glycation, or fatty acid load. Albumins with more reduced cysteine-34 content showed lower pro-inflammatory activity, but this correlation was lost when controlling for endotoxin levels, suggesting confounding by endotoxin contamination. Direct testing of isolated albumin redox fractions and in vitro glycated albumins confirmed that neither oxidation nor glycation of albumin, in the absence of endotoxin, triggered inflammatory responses in PBMCs. Inducing effects were found to be related to contaminating ligands and not to albumin itself. Our findings emphasize the need for rigorous product characterization and highlight the critical importance of endotoxin as a confounding factor in immune assays involving research grade albumin preparations.