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The plasma half-life of recombinant human interleukin-6 (rhIL-6) was determined in rats by measuring the disappearance of the biological activity as well as of the radioactivity of 125I-rhIL-6 from the circulation. The kinetics of clearance were biphasic. It consisted of a rapid initial disappearance corresponding to a half-life of 3 min, and of a second slow one corresponding to a half-life of about 55 min. By cellulose-acetate electrophoresis it was shown that rhIL-6 binds to a plasma protein resulting in a complex migrating in the β-γ region; 20 min after intravenous injection, about 80% of the 125I-rhIL-6 that had disappeared from the circulation was found in the liver. 125I-rhIL-6 was exclusively localized on the surface of parenchymal cells suggesting the existence of an interleukin-6 receptor on the hepatocytes. [ABSTRACT FROM AUTHOR]

Secretory products of cultured human blood monocytes contain a hepatocyte-stimulating factor which is able to induce the acute-phase proteins α2-macroglobulin and fibrinogen in rat liver cells. Total RNA was isolated from unstimulated and lipopolysaccharide-stimulated human monocytes and translated in a reticulocyte lysate. The capability of the cell-free synthesized proteins to induce the acute-phase proteins a2-macroglobulin and fibrinogen was assayed in rat hepatocyte primary cultures and in the rat hepatoma cell line Fao. The products translated from the mRNA of lipopolysaccharide-stimulated human monocytes induced mRNAs for α2-macroglobulin and fibrinogen and therefore contain hepatocyte-stimulating factor. The translation products of unstimulated monocytes had no effect. A cDNA containing the coding sequence for interleukin-6 (B-cell stimulatory factor 2, interferon-β2/26-kDa protein, interleukin HP1) derived from human T-cells cloned into the transcription vector pGEM4 was transcribed in vitro. Translation of the isolated RNA in a reticulocyte lysate led to the synthesis of a protein of about 25 kDa. This cell-free synthesized interleukin-6 exhibited hepatocyte-stimulating activity measured by the induction of β-fibrinogen mRNA in Fao cells. Using an antibody against interleukin-6, two proteins of 22 kDa and 23 kDa were immunoprecipitated from the culture medium of lipopolysaccharide-stimulated human monocytes. These two proteins were not synthesized by unstimulated monocytes. When total RNA from unstimulated human monocytes and lipopolysaccharide-stimulated human monocytes and lymphocytes was subjected to Northern analysis and hybridized with the interleukin-6 cDNA, a strong hybridization signal corresponding to an RNA of about 1300 bases was detected only in the RNA from lipopolysaccharide-stimulated human monocytes, indicating that human monocytes express the interleukin-6 gene after stimulation. The data presented in this paper strongly suggest that hepatocyte-stimulating factor from human monocytes and interleukin-6 from T-cells are identical. [ABSTRACT FROM AUTHOR]

The regulation of the three major acute-phase proteins α2-macroglobulin, cysteine proteinase inhibitor and α1-antitrypsin by recombinant human interleukin-1β, recombinant human interleukin-6 and recombinant human tumor necrosis factor α was studied in rat hepatocyte primary cultures. Synthesis and secretion of the acute-phase proteins was measured after labeling with [35S]methionine and immunoprecipitation. Incubation of hepatocytes with interleukin-6 led to dose-dependent and time-dependent changes in the synthesis of the three major acute-phase proteus and albumin, similar to those occurring m vivo during experimental inflammation. α2-Macroglobulin and cysteine proteinase inhibitor synthesis was induced 54-fold and 8-fold, respectively, 24 h after the addition of 100 units/ml interleukin-6. At the same time synthesis of the negative acute-phase protein albumin was reduced to 30% of controls. Half-maximal effects were achieved with 4 units interleukin-6/ml. Interleukin-lβ bad only a partial effect on the regulation of the fore proteins studied: only a twofold stimulation of α2-macroglobulin and a 60% reduction of albumin synthesis were observed. Tumor necrosis factor α did not alter the synthesis of acute-phase proteins. The stimulation of α2-macroglobulin and cysteine proteinase inhibitor synthesis by interleukin-6 was inhibited by interleukin-lβ in a dose-dependent manner. In pulse-chase experiments the effect of interleukin-lβ, interleukin-6 and tumor necrosis factor a on the secretion of acute-phase proteins was examined. Interleukin-6 markedly accelerated the secretion of total proteins and α2-macroglobulin, whereas the secretion of cysteine proteinase inhibitor, α1-antitrypsin and albumin was not affected. The inhibition of N-glycosylation by tunicamycin abolished the effect of interleukin-6 on the secretion of α2-macroglobulin, indicating a possible role of interleukin-6 on N-glycosylation. [ABSTRACT FROM AUTHOR]

The biosynthesis and secretion of α1-antitrypsin was studied in rat hepatocyte primary cultures. After labeling with [35S]methionine an α1-antitrypsin with an apparent molecular weight of 49000 estimated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis was immunoprecipitated from the cell homogenate. This intracellular form of α1 -antitrypsin could be deglycosylated by endoglycosidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. Pulse-chase experiments showed that about 30 min after its synthesis the transformation of the 49000-Mr α1 -antitrypsin to a protein with an apparent molecular weight of 54000 began. Only this 54000-Mr protein was secreted by the hepatocytes. The 54000-Mr α1 -antitrypsin was not sensitive to endoglycosidase H, but sensitive to neuraminidase, and it incorporated [³H]galactose and [³H]fucose indicating that its oligosaccharide chains were of the complex type. In the presence of tunicamycin, which blocks the formation of N-asparagine-linked oligosaccharide chains, an unglycosylated α1-antitrypsin with an apparent molecular weight of 41000 was found in the cells as well as in the medium. However, tunicamycin decreased the secretion of α1-antitrypsin by 60–70%, whereas the secretion of albumin remained unaffected. In the presence of colchicine the secretion of both α1-antitrypsin and albumin was impaired. The results demonstrate the importance of glycosylation for the secretion of α1-antitrypsin. [ABSTRACT FROM AUTHOR]

The biosynthesis of α1-antitrypsin, a secretory glycoprotein, has been studied in vitro in a cell-free translation system from wheat germ and in vivo with hepatocytes. When messenger RNA from rat liver polysomes was translated in a heterologous cell-free system a translation product was immunoprecipitated by antie(α1antitrypsin) which had a smaller molecular weight (42000) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis than the authentic glycoprotein (54000) isolated from rat serum. An intact amino terminus of the α1-antitrypsin synthesized in vitro could be demonstrated by repetitive Edman degradation. When hepatocytes, labeled with [35S]methionine. were analyzed with respect to newly synthesized α1-antitrypsin, a protein with a molecular weight of 49000 was immunoprecipitated from a high-speed supernatant. When the hepatocytes were treated with tunicamycin, an inhibitor of protein glycosylation, a protein of a molecular weight of 40000 was immunoprecipitated from the cells as well as from the medium. Interestingly, an α1-anti- trypsin form of an even higher molecular weight (54000) was immunoprecipitated from the hepatocyte monolayer medium. Several conclusions can be drawn from these findings. First, α1-antitrypsin is synthesized as a larger- molecular-weight precursor with an extra amino-terminal sequence of about 20 amino acids. Second, the liver form of α1-antitrypsin is different from the α1-antitrypsin which has been secreted, and third, the carbohydrate moieties of rat liver α1-antitrypsin do not appear to represent an absolute requirement for the secretion process. A length of 1930 ± 170 nucleotides was estimated for the poly(A)-rich mRNA of the precursor of rat liver α1-antitrypsin; a non-coding region of about 700 nucleotides must exist. Furthermore, it is shown that the major part of the poly(A)-rich RNA for α1-antitrypsin is found in loosely and tightly membrane-bound polyribosomes. This finding is in agreement with the current ideas on the site of synthesis of secretory proteins. [ABSTRACT FROM AUTHOR]

Rat α1-inhibitor 3 clones were isolated by immunological screening of a λgt11 cDNA library prepared from rat liver poly(A)-rich RNA. The recombinant cDNA clones were identified by the absence of their immunoprecipitable products following hybrid-arrested in vitro translation. The size of the cognate poly(A)-rich RNA was estimated to be roughly 5000 residues. Approximately 16 h after induction of inflammation the amount of or α1-inhibitor 3 poly(A)-rich RNA decreases as shown by dot-blot hybridization and Northern analyses. The response of this negative acute-phase plasma protein to inflammation may therefore be considered to be at the pretranslational level. The characterized DNA constitutes an open reading frame of 225 amino acids followed by a canonical eucaryotic polyadenylation signal and a poly(A) tail. Sequence microheterogeneity, particularly in the 3′-flanking region was observed. An amino acid homology of 70% for α1-inhibitor 3 with human and rodent α2-macroglobulin emphasizes the evolutionary relationship of the macroglobulins. [ABSTRACT FROM AUTHOR]