Serum Lipocalin-2 (LCN-2) as a major acute phase protein under different pathological conditions in vivo and in vitro studies

Lipocalin-2 (LCN-2) is a pleiotropic 25-kDa secretory protein, currently used as a biomarker for renal injury and inflammation. Its serum level is increased under different pathological conditions but the source and cause are unclear. The aim of our study was to prospectively evaluate the LCN-2 expression in different pathological conditions. The objective of our study was to determine LCN-2 expression in a rat and mouse model of sterile abscess and in a model of rat liver and lung exposed to single dose x-irradiation as oxidative stress is induced in both models. The current study compares LCN-2 gene expression with known major acute phase proteins in the liver in a rat and mouse model of turpentine-oil (TO)- induced sterile abscess. Furthermore, it shows that serum Lipocalin-2 is a potential Biomarker of radiation damage of liver but not lung. Serum LCN-2 concentrations increased dramatically up to 200-fold (20 µg/ml) at 48h after TO-injection. A strong elevation of LCN-2 mRNA in rat liver was observed starting from 4h up to 48h after injection, with a maximum (8738±2104-fold) at 24h, which was further confirmed by Western blot analysis. In contrast, the increases in gene expression of α2-macroglobulin (α2M), the major acute phase protein and hemoxygenase-1 (HO-1), a positive acute phase protein were only 1025±505 and 47±12-fold respectively during acute-phase- response. No considerable change was observed in LCN-2 mRNA in rat kidney and other organs as compared to liver. Using the IL-6 knockout mice model, wild type mice showed a strong LCN-2 expression, with a maximum of 2498±84-fold in the liver, which is similar to that for serum-amyloid-A (SAA) (2825±233-fold), a major mouse acute phase protein. However such an increase was significantly inhibited in IL-6ko mice during APR. IL-6 treated rat hepatocytes induced a significant time dependent up-regulation of LCN-2, indicating that LCN-2 is active on the executive side of the acute phase response, which is induced by IL-6. Also in our second model of acute phase reaction, LCN-2 serum levels increased significantly (up to 2.5 fold) within 24 hours after direct liver irradiation. No increase in serum levels were detected lung irradiation. LCN-2 specific transcripts increased significantly up to 552 ±109-fold at 24h after liver irradiation which was further confirmed by western blot analysis. Immunohistology of the liver detected positivity in recruited granulocytes within 1 hour after irradiation around central and portal fields. LCN-2 mRNA level of lung tissue showed an increased expression at 24 hours (9 ±2.3-fold) which was further confirmed at protein level by Western blot analysis. Lung immunohistology showed a high constitutive expression due to the high number of granulocytes. Irradiated hepatocytes showed higher LCN-2 expression as compared to myofibroblasts and Kupffer cells. Cytokine treatment specially IL-1β further increased LCN-2 gene expression in cultured hepatocytes. The current study compares LCN2 gene expression with known major acute phase proteins in the liver in a rat and mouse model of turpentine-oil (TO)- induced sterile abscess. LCN-2 is the major acute-phase protein as compared to α2M and HO-1 in rat and comparable with SAA in mouse. The gene expression is mainly controlled by IL-6. The liver is the main source of serum LCN-2 in the case of different acute-phase-responses supporting our earlier finding, the hepatocytes and not the granulocytes are the source for LCN-2 production in the liver and increased serum levels of LCN-2 in acute-phase-response. Single dose liver irradiation, but not lung irradiation induces a fast and significant increase of LCN-2 serum level. LCN-2 may be a suitable biomarker not only in acute-phase-response induced in TO model but also to determine the irradiated liver volume retrospectively in case of accidental liver irradiation to avoid RILD.