Abstract

Background: Albumin has been shown to modulate systemic inflammation in patients with decompensated liver cirrhosis receiving infusions of this protein as therapy. Although the anti-inflammatory effect of albumin has been described to be secondary to its internalization into mononuclear leukocytes, the cell type and the mechanism by which albumin is internalized by these immune cells are at present unknown.

Methods: The internalization of tetramethylrhodamine isothiocyanate (TRITC)-labelled albumin by B and T lymphocytes and different subsets of monocytes and dendritic cells was scrutinized by flow cytometry using the expression of distinct surface markers. The albumin-binding receptor(s) was identified by albumin immunoprecipitation and untargeted proteomics. Albumin uptake and intracellular trafficking in primary monocytes was examined by confocal microscopy and the endocytic routes involved in the internalization of TRITC-albumin were screened in THP-1 cells transduced with shRNA lentivirus blocking different key cellular endocytic pathways.

Results: Albumin was predominantly endocytosed and intracellularly accumulated by classical monocytes, which are highly phagocytic. In these cells and during the first 30 min after incubation, albumin was mainly located in early EEA1-positive endosomes whereas at later time points the albumin signal was also located in LAMP1-positive lysosomes. The shRNA lentivirus screening showed that albumin internalization was dependent on dynamin 2, ARF6 and ARF1, which are clathrin-independent routes. Untargeted proteomics of immunoprecipitated albumin identified two transmembrane proteins, the neonatal Fc receptor (FcRn) and CD44, as potential albumin receptor candidates. Of note, unlike THP-1 cells, classical monocytes freshly isolated from blood donors tested positive for intracellular albumin signal, indicating that the internalization of albumin by immune cells is a common occurrence in humans.

Conclusion: These findings offer new perspectives for the understanding of the mechanisms involved in the interaction of the albumin molecule with the immune system in those conditions where this protein is therapeutically used, as is the case of decompensated cirrhosis liver patients.