Isolation and characterization of a novel liver-derived immunoinhibitory factor.
Cytosolic extracts prepared from perfused whole liver or purified hepatocytes of C57BL/6 mice inhibited interleukin-2--and concanavalin A--induced spleen cell proliferation in vitro. In contrast, cytosolic extracts from purified nonparenchymal liver cells had no effect. Arginase and very-low-density lipoprotein were previously identified as two immuninhibitory substances present in liver cytosolic extracts. We demonstrated, however, that inhibitory activity remained after removal of very-low-density lipoprotein and arginase from liver cytosolic extract by repeated ultracentrifugation and gel filtration chromatography, respectively, suggesting the presence of another inhibitor. Further purification by anion-exchange chromatography and chromatofocusing led to the isolation of a novel liver-derived immunohibitory factor. This liver-derived immunoinhibitory factor is sensitive to pronase digestion and heat and acid treatment; it has an estimated isoelectric point of 8.25. The Mr of liver-derived immunoinhibitory factor is 28 kD as estimated from its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is identical under both reducing and nonreducing conditions, indicating a monomeric nature of this protein. Amino acid composition analysis discloses that liver-derived immunoinhibitory factor is relatively rich in glycine and proline residues. Interleukin-2--induced spleen cell proliferation in vitro is inhibited by ths liver-derived immunoinhibitory factor, with a 50% inhibitory dose of 1.4 nmol/L. Furthermore, the biological activity of the liver-derived immunoinhibitory factor is not confined to mouse spleen cells, since the growth of B16 mouse melanoma and H35 rat hepatoma cells is also inhibited. A comparison with other liver-derived immunoinhibitors reported previously supports our claim that the liver-derived immunoinhibitory factor is a novel inhibitory protein.
Department of Medicine, Veterans Administration Medical Center Sciences, Buffalo, NY 14215.
S P
SP
Tzung
K C
KC
Gaines
M
M
Henderson
T J
TJ
Smith
S A
SA
Cohen
D000818:Animals; D001119:Arginase / Q000737:chemistry / Q000302:isolation & purification*; D002455:Cell Division / Q000187:drug effects; D003600:Cytosol / Q000737:chemistry; D005260:Female; D006133:Growth Substances / Q000737:chemistry / Q000302:isolation & purification; D008099:Liver / Q000737:chemistry / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D014020:Tissue Extracts / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology
D006133:Growth Substances; D014020:Tissue Extracts; D001119:Arginase