A liver-derived immunosuppressive factor is an arginase: identification and mechanism of immunosuppression.

We found a substance in culture medium of neonatal pig liver fragments, which suppresses an immune response monitored by (3)H-thymidine incorporation using phytohemagglutinin (PHA)-stimulated lymphocytes. We named it as an immunosuppressive factor (ISF). To purify ISF, ammonium sulfate fractionation, DE52, SP-Sephadex, hydroxyapatite, blue Sepharose, heparin Sepharose and Superdex gel filtration columns were used. Using these purification procedures, ISF was purified 1,254-fold, with 9.2% recovery, from the culture medium of neonatal pig liver fragments, and was identified as arginase by its biochemical characteristics including molecular size, amino acid sequences of digested peptides and expression of arginase activity. The addition of ISF caused to decrease in arginine concentration in culture medium and at the same time DNA synthesis was suppressed dose-dependently, both of which were recovered by the addition of NOHA (N(G)-hydroxy-L-arginine), an arginase inhibitor. In addition, the depletion of arginine in culture medium also led to the inhibition of DNA synthesis. These results led us to the conclusion that immunosuppressive effect of ISF was due to arginase activity that decreased arginine concentration in culture medium, not to another function of ISF.

Hepatocyte proliferation factors from neonatal pig liver: purification and characterization.

Two factors were found in the condition medium of neonatal pig liver fragments, which were capable of stimulating DNA synthesis in primary hepatocytes. They were named hepatocyte proliferation factor (HPF)-1 and HPF-2 and purified 1,025- and 2,580-fold, respectively. Both HPF-1 and HPF-2 seem to be anionic at pH 8.0 judged from the elution pattern of DEAE (DE52) column chromatography. HPF-1 was recovered as a non-adsorbed fraction in blue Sepharose and heparin Sepharose columns, and had a molecular weight of 26-31 kDa as estimated by gel filtration in high salt condition. Purified HPF-1 stimulated DNA synthesis of primary rat hepatocytes, but suppressed that of HepG2 cells. HPF-2 strongly bound to blue Sepharose and heparin Sepharose columns, and had a molecular weight of 71-90 kDa as estimated by SDS-PAGE under non-reduced condition. Purified HPF-2 stimulated DNA synthesis of primary rat hepatocytes dose dependently but did not suppress that of HepG2 cells. From further biological and chemical characteristics studied in this paper, HPF-1 and HPF-2 may be novel stimulating proteins for hepatocyte proliferation, although the possibility that they are already known growth factors can not be excluded without complete purification and its cloning.