The acute-phase induction of alpha 2-macroglobulin in rat hepatocyte primary cultures: action of a hepatocyte-stimulating factor, triiodothyronine and dexamethasone

During inflammation a number of liver-derived plasma proteins increases in concentration. In the rat these so-called acute-phase proteins are mainly proteinase inhibitors, such as alpha 1-proteinase inhibitor, alpha 1-acute-phase globulin and alpha 2-macroglobulin. At present, the mechanisms responsible for the enhanced synthesis of acute-phase proteins are poorly understood. Therefore, we have studied the induction of alpha 2-macroglobulin synthesis in rat hepatocyte primary cultures. Adrenaline, triiodothyronine, estradiol and progesterone were tested for their ability to stimulate alpha 2-macroglobulin synthesis. Only triiodothyronine induced alpha 2-macroglobulin synthesis markedly. However, the presence of dexamethasone was a prerequisite for alpha 2-macroglobulin induction indicating a permissive action of glucocorticoids. Besides glucocorticoids and triiodothyronine a non-dialyzable factor (HSF) derived from rat Kupffer cells or human peripheral blood monocytes was found to be able to stimulate alpha 2-macroglobulin synthesis in hepatocytes. Equal amounts of HSF activity were found in conditioned media from lipopolysaccharide-stimulated and unstimulated rat Kupffer cells as well as in human monocytes. Since the supernatants of unstimulated rat Kupffer cells or human monocytes did not exhibit interleukin 1 activity, HSF activity distinct from interleukin 1 must exist. No HSF activity was found in media conditioned by rat Kupffer cells which had been treated with dexamethasone. Hepatocyte primary cultures were incubated with [35S]methionine-labeled proteins secreted by rat Kupffer cells. A 30 kDa polypeptide was found to be bound to or internalized by rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant human B cell stimulatory factor 2 (BSF-2/IFN-beta 2) regulates beta-fibrinogen and albumin mRNA levels in Fao-9 cells

Conditioned medium from human monocytes contains a partially characterized hepatocyte-stimulating factor that simultaneously elevates the mRNA levels of the acute-phase protein beta-fibrinogen and decreases albumin mRNA in rat hepatoma cells. We demonstrate that recombinant human B-cell stimulatory factor 2, which is identical to interferon-beta 2/26 kDa protein and interleukin-HP1, exhibits the same activity as hepatocyte-stimulating factor. Furthermore, a specific antibody against B-cell stimulatory factor 2 was able to inhibit hepatocyte-stimulating factor in conditioned medium from human monocytes. Our data show that hepatocyte-stimulating factor and B-cell stimulatory factor 2 are functionally and immunologically related proteins.     

Regulation of synthesis and secretion of major rat acute-phase proteins by recombinant human interleukin-6 (BSF-2/IL-6) in hepatocyte primary cultures

The regulation of the three major acute-phase proteins alpha 2-macroglobulin, cysteine proteinase inhibitor and alpha 1-antitrypsin by recombinant human interleukin-1 beta, recombinant human interleukin-6 and recombinant human tumor necrosis factor alpha 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 proteins and albumin, similar to those occurring in vivo during experimental inflammation. alpha 2-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-1 beta had only a partial effect on the regulation of the four patients studied: only a twofold stimulation of alpha 2-macroglobulin and a 60% reduction of albumin synthesis were observed. Tumor necrosis factor alpha did not alter the synthesis of acute-phase proteins. The stimulation of alpha 2-macroglobulin and cysteine proteinase inhibitor synthesis by interleukin-6 was inhibited by interleukin-1 beta in a dose-dependent manner. In pulse-chase experiments the effect of interleukin-1 beta, interleukin-6 and tumor necrosis factor alpha on the secretion of acute-phase proteins was examined. Interleukin-6 markedly accelerated the secretion of total proteins and alpha 2-macroglobulin, whereas the secretion of cysteine proteinase inhibitor, alpha 1-antitrypsin and albumin was not affected. The inhibition of N-glycosylation by tunicamycin abolished the effect of interleukin-6 on the secretion of alpha 2-macroglobulin, indicating a possible role of interleukin-6 on N-glycosylation.     

The acute-phase response of cultured rat hepatocytes. System characterization and the effect of human cytokines.

Hepatocytes were isolated from adult livers and cultured for periods of up to 5 days as monolayers at an initial density of 10(6) cells/10cm2 in Williams E medium containing insulin, dexamethasone and 5% foetal-calf serum. The daily production of 11 plasma proteins was measured by electroimmunoassay and compared with the concentrations of the same proteins in the plasma of normal rats and of those with experimental inflammation. Hepatocytes from normal rats synthesized proteins in relative amounts which were similar to the relative proportions of the same proteins in the plasma of turpentine-injected animals. The pattern changed only slowly during 5 days in culture, but it did so profoundly either when the medium was devoid of dexamethasone or when human cytokines (from endotoxin-stimulated monocytes or unstimulated human squamous-carcinoma cell line COLO-16) were added. The cytokines consistently increased the synthesis of alpha 2-macroglobulin and fibrinogen and depressed that of albumin; variable increases in the synthesis of alpha 1-acute-phase globulin, alpha 1-acid glycoprotein, haptoglobin and alpha 1-proteinase inhibitor, and variable decreases in transferrin synthesis, were seen, whereas the synthesis of antithrombin III, alpha 1-macroglobulin and prothrombin remained virtually unaffected. The cytokine effects on protein synthesis required the presence of dexamethasone. The hepatocyte-stimulating activity derived from monocytes chromatographed on Sephadex G-100 corresponding to 30 000 Da, as opposed to the lymphocyte-activating factor, which was eluted as a molecule of approx. 15 000 Da. This suggests that both activities probably reside with distinct molecular species in the preparations of human cytokines.     

Studies on the effect of the hepatocyte-stimulating factor on galactose-beta 1----4-N-acetylglucosamine alpha 2----6-sialyltransferase in cultured hepatocytes

Rat hepatic Gal beta 1----4GlcNAc alpha 2----6 sialyltransferase is released into the blood at elevated levels following an inflammatory challenge: this is a typical response of the group of plasma proteins known as acute-phase reactants. In the present study, primary cultures of liver parenchymal cells are used to demonstrate that the same hepatic cell type that produces plasma proteins such as fibrinogen also produces and releases sialyltransferase. Hepatic production of sialyltransferase is stimulated by a major regulator of hepatic acute-phase reactant production, the hepatocyte-stimulating factor (HSF), while another monokine, interleukin-1, does not affect hepatocyte sialyltransferase production. The maximum increase in sialyltransferase occurs 48 h after exposure to HSF which is considerably later than the fibrinogen response. The sialyltransferase that is stimulated by HSF is the Gal beta 1----4GlcNAc alpha 2----6 isozyme.     

Ciliary neurotrophic factor induces acute-phase protein expression in hepatocytes.

During inflammatory states, hepatocytes are induced to synthesize and secrete a group of proteins called acute-phase proteins. It has recently been shown that besides interleukin-6 (IL-6), related cytokines such as leukemia inhibitory factor, oncostation M and interleukin-11 are also mediators of the hepatic acute-phase response. All these mediators belong to the hematopoietic family of alpha-helical cytokines. Here we show that an additional member of this cytokine family, ciliary neurotrophic factor (CNTF), induces the hepatic acute-phase protein genes haptoglobin, alpha 1-antichymotrypsin, alpha 2-macroglobulin and beta-fibrinogen in human hepatoma cells (HepG2) and in primary rat hepatocytes with a time course and dose-response comparable with that of IL-6. Our next aim was to define the receptor components used by CNTF on hepatic cells. Using a cell-free binding assay we exclude that CNTF binds to the 80 kDa IL-6 receptor, a protein with significant homology to the CNTF receptor which has recently been cloned from neuroblastoma cells. In human hepatoma cells (Hep3B) which lack the leukemia inhibitory factor receptor, CNTF was not able to induce acute-phase protein synthesis, indicating that this receptor protein may be part of the functional CNTF receptor on hepatic cells.     

Interaction among hepatocyte-stimulating factors, interleukin 1, and glucocorticoids for regulation of acute phase plasma proteins in human hepatoma (HepG2) cells

Human hepatoma (HepG2) cells respond to unfractionated conditioned media of human squamous carcinoma (COLO-16) cells and lipopolysaccharide-stimulated human peripheral blood monocytes by increasing the synthesis of alpha 1-acid glycoprotein, haptoglobin, complement C3, alpha 1-antichymotrypsin, alpha 1-antitrypsin, and fibrinogen, while decreasing the synthesis of albumin. The regulation of the acute phase proteins is mediated by hepatocyte-stimulating factors (HSF) and interleukin 1 (IL-1) present in the conditioned medium. Purified HSF-I from COLO-16 cells stimulates preferentially alpha 1-acid glycoprotein synthesis, whereas COLO-HSF-II stimulates preferentially the synthesis of haptoglobin, fibrinogen, and alpha 1-antitrypsin. HSF from monocytes, which has been identified as interferon-beta 2 (B cell stimulating factor-2), displayed the same activity as COLO-HSF-II. Dexamethasone alone had no effect on acute phase plasma protein synthesis but enhanced the response to various HSF severalfold. IL-1 had a relatively low stimulatory activity on the synthesis of alpha 1-acid glycoprotein, haptoglobin, and alpha 1-antichymotrypsin but strongly reduced the basal expression of fibrinogen. The only synergistic action between IL-1 and HSF (or interferon-beta 2) was noted for the synthesis of alpha 1-acid glycoprotein. Tumor necrosis factor active on other hepatic cells failed to modulate significantly the expression of any plasma proteins in HepG2 cells. These studies showed that for an optimal HepG2-cell response a combination of HSF (or interferon-beta 2), IL-1, and dexamethasone is needed. This finding might indicate the identity of some of those hormones involved in regulation of the hepatic acute phase response in vivo.     

Distinct sets of acute phase plasma proteins are stimulated by separate human hepatocyte-stimulating factors and monokines in rat hepatoma cells

A subline of the rat hepatoma (H-35) cells has been identified which responds to hepatocyte-stimulating factors (HSFs) of human squamous carcinoma cells by increased synthesis of all major rat acute phase plasma proteins. The regulation occurs at the level of mRNA. Two HSFs (HSF-I and HSF-II) have been purified from conditioned medium of the squamous carcinoma cells. HSF-I is a protein with an Mr = 18,000 and pI 5.5, and HSF-II is a glycoprotein with an Mr = 34,000 and a broad, neutral to basic charge. In H-35 cells, HSF-I predominantly stimulates the synthesis of complement C3 and haptoglobin and acts synergistically with dexamethasone to stimulate alpha 1-acid glycoprotein. HSF-II stimulates cysteine protease inhibitor, alpha 1-antichymotrypsin, alpha 1-antitrypsin, fibrinogen, and hemopexin, and acts synergistically with dexamethasone to stimulate alpha 2-macroglobulin. Each HSF is between 10 and 100 times less effective in regulating proteins of the other set. Human tumor necrosis factor and interleukin-1 increase complement C3, haptoglobin, and alpha 1-acid glycoprotein, as does HSF-I, but are unable to modulate any of the other acute phase proteins. The monokines differ from HSF-I is their low activity in HepG2 cells and rat hepatocytes.     

Heterogeneous nature of the acute phase response. Differential regulation of human serum amyloid A, C-reactive protein, and other acute phase proteins by cytokines in Hep 3B cells

Because a number of different cytokines have been reported to regulate the synthesis of human, murine, and rat acute phase proteins (APP), we studied the effect of cytokines on production of several major human APP in a single system, the human hepatoma cell line Hep 3B. Conditioned medium (CM) prepared from human blood monocytes activated with LPS in the presence of dexamethasone led to substantial induction of serum amyloid A (SAA) and C-reactive protein (CRP) synthesis whereas the defined cytokines IL-1 beta, TNF alpha, and medium from a human keratinocyte cell line (COLO-16), containing hepatocyte-stimulating factor activity, failed to induce these two major APP. Induction of SAA and CRP was accompanied by an increase in concentration of their specific mRNA. Size fractionation of CM from activated monocytes by fast protein liquid chromatography indicated that SAA- and CRP-inducing activity eluted as a single peak with a Mr of approximately 18 kDa. alpha 1-Antitrypsin, which also failed to respond to IL-1 beta or TNF alpha, was induced by both CM and medium from COLO-16 cells. The induction of AT by CM was accompanied by an increase in specific mRNA. Induction of ceruloplasmin and alpha 1-antichymotrypsin and decrease in the synthesis of albumin was achieved by both CM and IL-1 beta. Ceruloplasmin and albumin responded in a comparable fashion to both TNF alpha and medium from COLO-16 cells; the response of ACT to these cytokines was not evaluated. These results indicate that human SAA and CRP are induced in Hep 3B cells by products of activated monocytes but not by IL-1 beta, TNF-alpha, or some hepatocyte-stimulating factor preparations and that a group of heterogeneous mechanisms are involved in the induction of the various human APP.     

Isolation and purification of rat acute-phase alpha 2-macroglobulin

Acute-phase alpha 2-macroglobulin was highly purified from the serum of rats in which this protein had been induced 48 h previously by the injection of croton oil, an inflammatory agent. The isolation protocol involved two non-denaturing steps; first, separation according to molecular weight by gel filtration on Ultrogel AcA 22 and second, negative affinity chromatography which bound contaminating proteins to the column while allowing acute-phase alpha 2-macroglobulin to pass through. Several criteria were used to assess the purity of acute-phase alpha 2-macroglobulin, after which the protein by mass determination and by two different protein assays. Pure rat acute-phase alpha 2-macroglobulin was used to produce a monospecific antiserum and to calibrate a secondary standard of rat acute-phase serum by developing and characterizing rocket immunoelectrophoresis assay.     

Stimulation of peripheral blood mononuclear cells with lipopolysaccharide induces expression of the plasma protein alpha2-macroglobulin

The human alpha(2)-macroglobulin gene is approximately 48 kb in size and consists of 36 exons, which encode the 180 kDa subunit of this large tetrameric protein. In this investigation, a procedure of sequencing human alpha(2)-macroglobulin mRNA, using mRNA from lipopolysaccharide-stimulated peripheral blood mononuclear cells as template in RT-PCR, was developed. Incubation of peripheral blood mononuclear cell populations with lipopolysaccharide induced alpha(2)-macroglobulin mRNA expression reaching levels detectable by RT-PCR. Extracted human alpha(2)-macroglobulin mRNA was used to determine the nucleotide sequence of a 500 bp DNA segment encoding the most C-terminal, receptor-binding part of the protein, using alpha(2)-macroglobulin specific primers. The sequence obtained matched the earlier published sequence of human alpha(2)-macroglobulin, except for three point mutations, i.e., cytosine for guanine, cytosine for thymidine and thymidine for adenine substitutions at positions 4369, 4423, and 4511, respectively. None of these alterations, however, affect the amino acid sequence of the protein. In conclusion, we demonstrate a new, improved, approach to sequence human alpha(2)-macroglobulin mRNA by overexpressing the protein in peripheral blood mononuclear cells. This procedure may be useful in the search for mutations in alpha(2)-macroglobulin, examining its role in the pathogenesis of human diseases.     

Long term production of acute-phase proteins by adult rat hepatocytes co-cultured with another liver cell type in serum-free medium

Three acute-phase proteins, haptoglobin, alpha 2-macroglobulin and hemopexin, as well as albumin, have been measured daily in the hydrocortisone-supplemented serum-free medium of pure and mixed cultures of adult rat hepatocytes for 5 and 20 days respectively. Whereas plasma protein production rapidly declined in pure culture, it remained relatively stable when hepatocytes were co-cultured with rat liver epithelial cells. In the latter cultures, an early stimulation of albumin and alpha 2-macroglobulin secretion was observed. In addition, four other plasma proteins, fibrinogen, alpha 1-acute-phase protein, alpha 1-acid glycoprotein and alpha 1-antitrypsin were shown by immunodiffusion to still be produced by day 20 of co-culture. These results suggest that hepatocyte co-cultures represent a suitable model for studying the mechanism which controls synthesis of plasma proteins, including acute-phase proteins by liver cells.     

Synthesis of hemopexin and cysteine protease inhibitor is coordinately regulated by HSF-II and interferon-beta 2 in rat hepatoma cells

Rat hepatoma (H-35) cells respond to hepatocyte-stimulating factors by increased expression of major acute phase plasma proteins. The synthesis of hemopexin is stimulated 10-fold by either hepatocyte-stimulating factor-II of human squamous carcinoma cells or hepatocyte-stimulating factor/interferon-beta 2 of activated human blood monocytes. The hormone specificity, time course and dose-dependence of hemopexin regulation is closely correlated with that of cysteine protease inhibitor. The coordinate expression of hemopexin and other type II acute phase proteins suggests the existence of common molecular regulatory mechanisms.     

The development of rat alpha 2-macroglobulin. Studies in vivo and in cultured fetal rat hepatocytes

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.     

Radioimmunoassay of rat acute-phase alpha 2-macroglobulin

A double-antibody radioimmunoassay (RIA) to acute-phase alpha 2-macroglobulin was developed for the quantitation of this large macromolecule in physiological fluids. The primary receptor for the RIA was a monospecific antiserum to purified acute-phase alpha 2-macroglobulin which produced a high titre (7.5 . 10(6)) antibody with a strong affinity for rat acute-phase alpha 2-macroglobulin (Ka = 1.24 . 10(11)) as measured by Scatchard analysis. The validity of the assay was confirmed by specificity for rat alpha 2-macroglobulin measured in various physiological fluids as assessed by parallel dose-response curves; and accuracy, measured by the analytical recovery of alpha 2-macroglobulin by the RIA in serum (104 +/- 7%) and buffer (103 +/- 7%), and the correlation (R = 0.999) of measurements of acute-phase alpha 2-macroglobulin-containing samples measured in serum and buffer. Reference acute-phase serum measured by this RIA and by rocket immunoelectrophoresis were 98.6% in agreement. Radioimmunoassay sensitivity was estimated at less than 1.0 ng alpha 2-macroglobulin/ml, measured over a range of 0-160 ng. Precision was assessed by intraassay (2.99 +/- 0.97%) and interassay (8.76 +/- 2.64%) variation. Evaluation confirmed that quantitation of rat acute-phase alpha 2-macroglobulin by this RIA met the criteria of sensitivity, validity and precision.     

A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18.

The E6 protein of human papillomavirus types 16 and 18 (HPV-16 and HPV-18) can stably associate with the p53 protein in vitro. In the presence of rabbit reticulocyte lysate, this association leads to the specific degradation of p53 through the ubiquitin-dependent proteolysis system. We have examined the E6-p53 complex in more detail and have found that association of E6 with p53 is mediated by an additional cellular factor. This factor is present in rabbit reticulocyte lysate, primary human keratinocytes and in each of five human cell lines examined. The factor is designated E6-AP, for E6-associated protein, based on the observation that the E6 proteins of HPV-16 and 18 can form a stable complex with the factor in the absence of p53, whereas p53 association with the factor can be detected only in the presence of E6. Gel filtration and coprecipitation experiments indicate that E6-AP is a monomeric protein of approximately 100 kDa.     

Biochemical and biological analysis of human interleukin 6 expressed in rodent and primate cells.

The cDNA for human interleukin 6 (IL 6) was stably expressed at high levels in the three mammalian cell lines COS-7, PA317, and GH3 to yield IL 6 proteins of 25 to 27, 26, 22 to 24, and 23 kDa molecular mass. Both size and relative amounts of the recombinant IL 6 (rIL 6) species produced correspond to those of natural IL 6 secreted by LPS-stimulated monocytes. Oligosaccharide analysis of recombinant IL 6 utilizing tunicamycin and endoglycosidases revealed O- and N-linked glycosylation that is comparable to that of natural IL 6 derived from human monocytes and fibroblasts. IL 6 expressed in each of the three cell lines was phosphorylated similarly to the IL 6 produced in human monocytes and fibroblasts. IL 6 secreted by the three different cell lines have marked differences in specific biological activities. COS-7 IL 6 appeared to be 12-fold more active in its hybridoma growth factor activity than that made in PA317 or GH3 cells. In contrast, PA317 and GH3 IL 6 were 230 and 6.7 times more effective than COS-7 IL 6 in inducing Ig production in CESS cells. Also, PA317 and GH3 IL 6 were more effective than COS-7 IL 6 in inducing the acute-phase protein fibrinogen in human hepatocytes. The rIL 6 species exhibited no antiviral activity.