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This study investigated the ability of recombinant interleukin-2 (IL-2) to modulate the ability of peripheral blood mononuclear cells (PBMCs) to stimulate an acute phase protein response in isolated human hepatocytes. The effect of IL-2 on the production of tumour necrosis factor-alpha (TNF) and interleukin-6 (IL-6) by PBMCs isolated from patients with gastrointestinal cancer, multiple organ failure, and healthy controls was also studied. The ability of supernatants from IL-2-treated PBMCs to elicit an acute phase response in hepatocytes was then investigated. IL-2 had no effect on IL-6 or TNF production by PBMCs isolated from any group in the presence or absence of bacterial lipopolysaccharide (LPS). Despite this, preincubation of PBMCs with IL-2 significantly reduced the potential of LPS-stimulated PBMC supernatants to stimulate production of alpha1 antichymotrypsin, alpha1-acid glycoprotein, and C-reactive protein by hepatocytes. These observations were not due to a direct effect of IL-2 on hepatocyte acute phase protein production. These findings suggest that in this model IL-2 may modulate PBMC-induced acute phase protein production through an IL-6 and TNF-independent pathway.

Background: Acute phase proteins (APPs) are enhanced in end-stage renal disease patients (ESRD) requiring dialysis treatment. They are involved in a variety of pathologic processes like muscle proteolysis, cachexia, regulation of appetite, and atherosclerosis. They are predictive for mortality. APPs are not only makers but also active substances. They are mainly produced in liver cells and are primarily, but not exclusively, regulated by proinflammatory cytokines. To what extent hepatic APPs are influenced by uremic toxins is still unclear. Therefore, we investigated the effects of different ultrafiltrates (UFs) on the synthesis of alpha1-acid glycoprotein (AGP) in HepG2 cells., Methods: A cross-sectional as well as a crossover study with high-/low-flux membranes was conducted to investigate the impact of UFs on bioactivity of liver cell cultures. Metabolic activity (MTT test), cytotoxicity (lactate dehydrogenase release), and the positive APP AGP were measured in HepG2 cells., Results: Cultured hepatocytes treated with UFs from high-flux membranes exhibited a higher cytotoxicity (18.6 +/- 0.3% high-flux vs. 13.9 +/- 0.2% low-flux, P < 0.001) and a lower metabolic activity (29.3% high-flux vs. 50.3% low-flux, P < 0.001) in comparison with low-flux UFs. In addition, enhanced APP secretion could be observed under costimulatory conditions (high-flux 5.0 +/- 0.7 vs. low-flux 3.1 +/- 0.6 ng/microg protein, P < 0.05). The effects of high- and low-flux UFs were strongly expressed at the beginning and were still significantly different after 120 minutes of hemodialysis (HD) treatment. The crossover experiments confirmed that UFs collected during high-flux HD had a higher capacity to stimulate AGP synthesis in liver cells., Conclusion: The effects of UFs from dialysis patients demonstrate that hepatotoxic substances can be removed by dialysis. Stimulating the acute phase response UF collected during high-flux HD had a higher impact on liver cells in comparison with low-flux UF. These substances are putative cofactors involved in cytokine regulation.

Responsiveness to cytokine-mediated acute inflammatory stimuli of the highly differentiated and polarized WIF-B hybrid cell line was studied by measuring the induction of alpha 1-acid glycoprotein and alpha 2-macroglobulin mRNAs after interleukin-1, interleukin-6 and tumor necrosis factor-alpha treatments in the presence of dexamethasone. Compared with their Fao parent, WIF-B cells were 10 times more responsive to 24-h interleukin-6 induction regarding alpha 2-macroglobulin induction. At variance from the response measured in Fao cells, the late effects of interleukin-6 treatment confirmed the higher sensitivity of WIF-B cells to this cytokine as a 72-h treatment as 10 times more effective than a 24-h treatment at inducting alpha 1-acid glycoprotein mRNA. These findings highlight the hepatocyte differentiation of WIF-B cells compared with other hepatoma cell lines, with respect to the regulation of acute-phase protein gene expression. They also make WIF-B cells a convenient model to study the molecular effects of interleukin-6 in terms of transduction and/or transcription, and the many cross-talks that occur during the regulation of acute-phase protein gene expression.

The gene encoding alpha-1-acid glycoprotein (AGP), one of the major acute-phase proteins, is positively controlled at the transcriptional level by cytokines (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) and glucocorticoids. Here, we show that growth hormone (GH) treatment of isolated rat hepatocytes in vitro reduces AGP messenger RNA (mRNA) expression. AGP gene expression remained inducible by IL-1, IL-6, and phenobarbital (PB) in GH-treated hepatocytes. Interestingly, the repressive effect of GH on AGP gene expression was also observed in vivo: liver AGP mRNA content was strongly increased in hypophysectomized rats, and GH treatment of these animals led to a decrease in mRNA to levels lower than those in untreated control animals. Moreover, the inhibitory effect of GH mainly occurs at the transcriptional level and can be observed as little as 0.5 hours after GH adding in vitro to isolated hepatocytes. These results show negative regulation of AGP gene expression and strongly suggest that GH is a major endogenous regulator of constitutive AGP gene expression. Moreover, transfection assays showed that the region of the AGP promoter located at position -147 to -123 is involved in AGP gene regulation by GH. Furthermore, GH deeply modifies the pattern of nuclear protein binding to this region. GH treatment of hypophysectomized rats led to the release of proteins of 42 to 45 and 80 kd and to the binding of proteins of 48 to 50 and 90 kd.

During the course of studies designed to identify the role of cytokines in the reprioritization of hepatic protein synthesis associated with cachexia we detected a hepatocyte-stimulating moiety in the supernatants of pancreatic cancer cells that was unrelated to interleukin (IL)-6. This study identifies that moiety as IL-8 and investigates the role of IL-8 in the induction of acute-phase protein production. The human pancreatic cancer cell line MIA PaCa-2 produced >1 ng/ml of IL-8 per 24 h, and supernatants from this cell line induced C-reactive protein (CRP) production from isolated human hepatocytes. Addition of neutralizing anti-human IL-8 antibody to such supernatants produced almost complete inhibition of CRP production. The addition of recombinant human IL-8 to hepatocytes resulted in a dose-dependent increase in CRP, alpha1-acid glycoprotein, and alpha1-antichymotrypsin production and a decrease in the production of transferrin and prealbumin. This study demonstrates that recombinant or tumor-derived IL-8 can modulate acute-phase protein production from isolated human hepatocytes and from human hepatoma cells.

The purpose of this study was to define the differences in heat shock protein (hsp)70, albumin, alpha(-1)-acid glycoprotein (AGP), and CCAAT enhancer binding proteins (C/EBP) alpha and beta mRNA between hepatic ischemia and reperfusion, and to begin to explore C/EBP protein production. These genes have been found important in the hepatic response to lipopolysaccharide and inflammation. In two experiments, Sprague-Dawley rats underwent temporary occlusion of the median and left hepatic lobe vasculature. The first experiment included a single sham-operated group and ligation of the right hepatic lobes during reperfusion. It compared 30 and 60 min ischemia to 2 h reperfusion. The second experiment included a sham-operated group for every time point, and the right hepatic lobes were not ligated during reperfusion; a 30-min ischemia group was compared to 2-, 5-, and 24-h reperfusion groups. Total RNA from the ischemic lobes was analyzed by Northern hybridization for hsp70, albumin, AGP, and C/EBPalpha and beta. C/EBPalpha and beta proteins were compared by Western blotting. Differences in experimental design played an important role in interpretation of results. hsp70 mRNA began to increase during ischemia. Albumin mRNA remained constant during ischemia and reperfusion. The ischemic hepatocyte nucleus is not quiescent and retains the ability to upregulate certain genes, e.g., hsp70. Changes in mRNA in response to hepatic ischemia/reperfusion occur rapidly. Hepatic ischemia/reperfusion does not recapitulate the classic acute phase response; albumin is not down regulated during reperfusion.

The mRNAs of the CCAAT/enhancer-binding trans-activator proteins (C/EBPalpha and C/EBPbeta) serve as templates for the differential translation of several isoforms which have specific transcriptional regulatory functions. By using an oligonucleotide corresponding to the C/EBP binding site of the mouse alpha1-acid glycoprotein promoter, we detected multiple forms of C/EBPalpha and C/EBP++ beta proteins in the mouse liver that have DNA-binding activity. By using specific antisera, we detected C/EBPalphas with molecular masses of 42, 38, 30, and 20 kDa that have DNA-binding activity. The pool levels of the 42- and 30-kDa isoforms were high in control nuclear extracts and decreased significantly after lipopolysaccharide (LPS) treatment. The binding activity and protein levels of the 20-kDa isoform are low in controls and increase dramatically after LPS treatment. C/EBPbeta isoforms with molecular masses of 35, 20, and 16 kDa were also detected. The 35-kDa pool level did not change whereas the 20-kDa isoform was strongly induced in response to LPS. Western (immunoblot) and Southwestern (DNA-protein) analyses show that p42 C/EBPalpha forms specific complexes with the alpha1-acid glycoprotein oligonucleotide in control nuclear extract and that p20 C/EBP beta forms complexes in LPS-treated liver. Our studies suggest that synthesis of specific C/EBPalpha and C/EBPbeta isoforms occurred in the normal liver in vivo and that LPS mediated a differential initiation and inhibition of translation at specific AUG sites within each mRNA. The qualitative and quantitative changes in C/EBPalpha and C/EBPbeta isoform pool levels suggest that LPS or an LPS-stimulated factor can regulate the selection of AUG start sites for both activation and repression of translation. This regulation appears to involve an LPS-mediated down-regulation of initiation at the first AUG codon of the 42-kDa C/EBPalpha and dramatic translational up-regulation at the fifth AUG codon of the 20-kDa C/EBPalpha and the third AUG codon of the 20-kDa C/EBPbeta. These regulatory events suggest the existence of proteins that may act as translational trans-acting factors.

While the effects of insulin, dexamethasone and cytokines on alpha 1-acid glycoprotein gene expression have been investigated in various hepatoma cell lines, the individual and combined effects of these components on the expression of this gene have been rarely studied in cultured normal rat hepatocytes. In this cell model, we have shown that mRNA levels of alpha 1-acid glycoprotein were not decreased at least during the first 24 h of culture under basal conditions. During these short-term cultures, the expression of alpha 1-acid glycoprotein in normal hepatocytes showed a high degree of responsiveness to dexamethasone alone (20-fold increase) and to dexamethasone associated with various cytokines (interleukin-1 beta, interleukin-6 and tumor necrosis factor alpha) with a 40 to 100-fold increase depending on the cytokine. Insulin alone did not modify alpha 1-acid glycoprotein mRNA; however, this hormone exerted a positive effect (about 50% increase) in the presence of dexamethasone or dexamethasone with cytokines. These results indicate that the regulation of alpha 1-acid glycoprotein in cultured normal rat hepatocytes presents major differences when compared to reported observations in rat hepatoma cell lines.

The serum level of rat alpha 1-acid glycoprotein is significantly increased by treatment with phenobarbital, and in vivo studies have shown that phenobarbital seems to act mainly at the transcriptional level. To show the direct mediating effect of phenobarbital on alpha 1-acid glycoprotein gene expression, we investigated the ability of primary cultured rat hepatocytes to respond to in vitro phenobarbital administration. Phenobarbital increased both alpha 1 acid glycoprotein secretion and corresponding mRNA levels in primary rat hepatocytes cultured on matrigel. Used in combination with interleukin-1, interleukin-6 and dexamethasone, phenobarbital had an additive or synergistic effect on alpha 1-acid glycoprotein synthesis. These results show that (a) phenobarbital acts directly on hepatocytes by increasing alpha 1-acid glycoprotein gene expression and (b) this effect is mediated by a specific mechanism independent of pathways involved in alpha 1-acid glycoprotein induction by interleukin-1, interleukin-6 and glucocorticoids.

Objective: Nitric oxide (NO) is a potent biologic mediator produced by hepatocytes following exposure to cytokines and lipopolysaccharide (LPS). These cytokines are also known to regulate induction of the hepatic acute-phase response. The objective of this study was to determine whether inducible nitric oxide synthase (iNOS), the enzyme that produces NO, is expressed as part of the hepatic acute-phase response., Design: The gene expression for inducible NOS (iNOS) as well as alpha 1-acid glycoprotein (AGP), an established acute-phase reactant, was measured by Northern blot analysis in rat hepatocytes in vivo during endotoxemia (LPS injection) and during the acute-phase response produced by hindlimb turpentine injection. Hepatocyte iNOS messenger RNA (mRNA) levels were correlated with iNOS activity and circulating plasma nitrite and nitrate levels. In vitro, iNOS and AGP mRNA levels were determined in cultured hepatocytes stimulated with interleukin 6 (IL-6), interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), or dexamethasone., Results: The AGP mRNA levels were increased in vivo following both LPS and turpentine injection, while iNOS expression was induced only by LPS injection. Hepatocyte iNOS activity and plasma nitrite and nitrate levels also increased after LPS treatment. In vitro, the cytokine combination IL-6, IL-1 beta, and TNF-alpha induced hepatocyte iNOS expression but had minimal effects on AGP in the absence of dexamethasone. Addition of dexamethasone alone markedly increased AGP mRNA levels, with further increases seen with TNF-alpha or IL-1 beta addition. In contrast, dexamethasone decreased iNOS expression., Conclusion: The results show that hepatocyte iNOS expression is not part of the acute-phase response induced by remote inflammation and indicates that iNOS is differentially regulated from the acute-phase reactant, AGP.

The synthesis of albumin and other hepatic proteins, many regulated as part of the acute phase response, is increased in the nephrotic syndrome. It has been postulated that synthesis of all proteins secreted by the liver is increased by the same mechanism in the nephrotic syndrome. However, the observation that synthesis of some apolipoproteins is not increased suggests that only a specific group of proteins may be similarly regulated in nephrosis. We measured synthesis of albumin and of two acute phase proteins, fibrinogen and alpha 1-acid glycoprotein (alpha 1-AG), in rats with Heymann nephritis (HN), their mRNA concentration in liver, and the rate of transcription of their genes by hepatic nuclei. Albumin and fibrinogen mRNA levels almost doubled in HN, but alpha 1-AG mRNA was unchanged. Ribosomal RNA (28S) concentration and transcription were also increased significantly in HN. Transcription of albumin and fibrinogen also increased twofold, but transcription of alpha 1-AG was unchanged. Fibrinogen and albumin synthesis each increased more than fourfold in HN and correlated with one another. In contrast alpha 1-AG synthesis only increased by 50% and did not correlate with albumin synthesis. Both albumin and alpha 1-AG were lost in the urine of HN, and their plasma concentrations were reduced. Fibrinogen was not lost in the urine and its plasma concentration was significantly increased in HN. Synthesis of a group of proteins including both positive acute phase (fibrinogen) and negative acute phase (albumin) proteins is increased transcriptionally in the nephrotic syndrome. Synthesis of other proteins is increased posttranscriptionally.(ABSTRACT TRUNCATED AT 250 WORDS)

The synthesis and secretion of several acute-phase proteins increases markedly following physiological stress. alpha 1-Acid glycoprotein (AGP), a major acute-phase reactant made by the liver, is strongly induced by inflammatory agents such as lipopolysaccharide (LPS). Nuclear run-on assay showed a 17-fold increase in the rate of AGP transcription 4 h following LPS injection. DNase I footprinting assays revealed multiple protein binding domains in the mouse AGP-1 promoter region. Region B (-104 to -91) is protected by a liver-enriched transcription factor that is heat labile and in limiting quantity. An adjacent region, C (-125 to -104), is well-protected by nuclear extracts from hepatocytes. Electrophoretic mobility shift assays indicated that only one DNA-protein complex can form with an oligonucleotide corresponding to region B. However, nuclear proteins from untreated mouse liver can form three strong complexes (C1, C2, and C3) and a weak one (C4) with oligonucleotide C. An acute-phase-inducible DNA-binding protein (AP-DBP) forms complex 4. A dramatic increase (over 11-fold) in AP-DBP binding activity is seen with nuclear proteins from LPS-stimulated animals. Interestingly, AP-DBP, a heat-stable factor, can form heterodimers with the transcription factor CCAAT/enhancer binding protein (C/EBP). Furthermore, purified C/EBP also binds avidly to region C. Our studies indicate that several liver-enriched nuclear factors can interact with AGP-1 promoter and that AP-DBP binds to the AGP-1 promoter with high affinity only during the acute-phase induction.

The serum level of alpha 1-acid glycoprotein (alpha 1-AGP) is significantly increased in various animal species by treatment with cytokines, glucocorticoids and phenobarbital. The mechanisms responsible for the cytokine-induced and glucocorticoid-induced increases are now well documented, but not so in the case of phenobarbital. The main purpose of this study was to assess whether phenobarbital acts on alpha 1-AGP synthesis in the liver at the transcriptional or translational level. Male Dark Agouti rats received 70 mg phenobarbital/kg daily for 7 days. The analysis of total hepatic RNA showed that a single injection of phenobarbital induced an 11-fold increase in phenobarbital-dependent cytochrome P450IIB mRNA, whereas seven injections of phenobarbital were required to induce a maximum 5.5-fold increase in alpha 1-AGP mRNA. Concurrently, the transcription rate of the alpha 1-AGP gene rose 3.5-fold. Hepatocytes isolated after the seventh injection of phenobarbital showed a threefold increased capacity to secrete alpha 1-AGP, corresponding to a 3.2-fold increased alpha 1-AGP mRNA content in the liver. In conditions in which its effect on the induction of alpha 1-AGP synthesis was maximum, phenobarbital caused a 30% reduction in liver albumin mRNA and in albumin secretion by isolated hepatocytes, resulting from a 60-70% reduction in the rate of transcription of the albumin gene measured in isolated nuclei. We conclude that the effect of phenobarbital on alpha 1-AGP and albumin gene expression occurs at the transcriptional rather than the translational level.

The effect of ischemia on hepatic protein synthesis during sepsis is not known, but is of clinical relevance, since hepatic blood flow decreases during the late phase of sepsis. In this study, synthesis of acute-phase proteins was measured in perfused livers of rats 16 hours after sham operation or cecal ligation and puncture. Livers from each group had 45 minutes of complete ischemia or control perfusion. Protein synthesis was measured during two hour perfusion after the ischemia or control period, by determining incorporation of 3H-leucine into total secreted trichloracetic acid precipitated proteins, immunoprecipitated complement component C3 and albumin and phosphotungstenate-precipitated alpha 1-acid glycoprotein. Lactate, glutamine-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels in the perfusate were measured during preischemic and postischemic perfusion. Tissue glutathione levels were measured at the end of the perfusion. Synthesis of alpha 1-acid glycoprotein was increased by 100 per cent and albumin synthesis decreased by 46 per cent in septic livers, consistent with an acute-phase response and apparent downregulation of albumin synthesis during early sepsis. Synthesis rates were reduced by 50 to 60 per cent after ischemia in perfused livers from sham operated rats and 70 to 80 per cent in livers from septic rats. Hepatic production of interleukin-1 was not different between the groups during perfusion. GOT and GPT levels increased significantly during ischemia of both nonseptic and septic livers and rapidly returned toward baseline during reperfusion. Lactate levels were higher in perfusate of septic than of nonseptic livers before ischemia and increased further during ischemia. The results suggest that ischemia inhibits production of secreted hepatic proteins similarly in nonseptic and septic livers, but perhaps to a slightly greater extent in septic livers.

The relative occurrence of genetic variants of human alpha 1-acid glycoprotein (AGP) in relation to changes in glycosylation was studied in sera of patients with burn injury, media of cytokine-treated primary cultures of human hepatocytes and Hep 3B cells, and sera of transgenic mice expressing the human AGP-A gene. It is concluded (i) that the glycosylation of AGP was not dependent on its genetic expression and (ii) that both the variants determined by the AGP-A gene as well as by the AGP-B/B' genes are increased after inflammation or treatment with interleukins 1 and 6.

The pattern of gene expression in fetal hepatocytes transformed in culture with a hepatocarcinogen (FRL cells) is studied with respect to a range of markers which are either developmentally regulated and/or shown to be expressed at high levels in hepatoma cells. The relative abundance of the respective mRNAs is determined and immunocytochemistry is used to detect the respective proteins in cultured cells. When compared with its normal counterpart, FRL cells retain the expression of transferrin, alpha 1-acid glycoprotein, gamma-glutamyltranspeptidase, and tyrosine aminotransferase at near normal levels, while expression of the liver-specific isoenzymes of pyruvate kinase (L form) and aldolase (B form) is reduced. The cell lines are different in that they fail to express albumin, alpha-fetoprotein, thiostatin and alpha 2-macroglobulin, and they express high levels of M2-pyruvate kinase and aldolase A, markers often found in abundance in hepatoma cells. Therefore transformation has resulted in different effects on different genes. Furthermore, it is of interest to find that the cells coexpress both forms of the pyruvate kinase isoenzymes which does not occur in the normal developing hepatocyte. These results indicate that it is possible to use this model to study changes which accompany transformation of fetal rat hepatocytes. The resulting cell lines have a stable phenotype and retain the changes which result from transformation even after extended passaging. This facilitates comparisons between the precursor cell and the tumor cell, both of which can be maintained under controlled conditions which exist in culture.

The effect of D-galactosamine on protein N-glycosylation was studied in rat hepatocyte primary cultures for alpha 1-antitrypsin (three complex type oligosaccharide chains) and alpha 1-acid glycoprotein (six complex type oligosaccharide chains). D-Galactosamine at a concentration of 4 mM inhibited partially de novo N-glycosylation leading to the formation of alpha 1-antitrypsin lacking one to two and of alpha 1-acid glycoprotein lacking one to five of its carbohydrate side chains. In addition D-galactosamine interfered with oligosaccharide processing, leading to the formation of some carbohydrate side chains remaining in an endoglucosaminidase H sensitive, i.e., not completely processed, form. D-Galactosamine impaired the secretion of alpha 1-antitrypsin and of alpha 1-acid glycoprotein but did not inhibit the secretion of the unglycosylated albumin. The inhibitory effect of D-galactosamine on de novo glycosylation as well as on oligosaccharide processing lasted for at least 24 h after it had been removed from the cells. D-Galactosamine impaired the glycosylation of alpha 1-antitrypsin only in hepatocytes, but not in human monocytes. Furthermore, D-galactosamine did not impair the N- and O-glycosylation of interleukin-6 in human monocytes and in MRC 5 fibroblasts. The results indicate that the effect of D-galactosamine on protein glycosylation is restricted to D-galactosamine metabolizing hepatocytes and is not exerted by the drug itself but by its metabolites.

The ability of the fetal rat to respond to interleukin 1 beta (IL1 beta) by expressing alpha 1-acid glycoprotein (AGP) was investigated. Eight and 20 h after injection of 7 ng IL1 beta into 19-day fetuses, liver AGP mRNA increased by a factor of 66 and 82 respectively, while serum AGP levels increased by a factor of 3 and 5. Similar treatment of the mothers altered in the fetuses neither AGP serum levels nor the amount of liver AGP mRNA. The induction of AGP gene expression in the fetal liver in response to IL1 beta was similar to that observed in the adult liver. These results demonstrate that at day 19 the fetal rat liver has acquired a mature acute-phase system.

Recombinant human IL-6 (rhIL-6) is a potent inducer of the synthesis of acute phase proteins in adult human hepatocytes. A wide spectrum of acute phase proteins is regulated by this mediator. After labeling of rhIL-6 stimulated human hepatocytes with [35S]methionine acute phase protein synthesis was measured by immunoprecipitation. Serum amyloid A, C-reactive protein, haptoglobin, alpha 1-antichymotrypsin and fibrinogen were strongly induced (26-, 23-, 8.6-, 4.6- and 3.8-fold increases, respectively). Moderate increases were found for alpha 1-antitrypsin (2.7-fold) and alpha 1-acid glycoprotein (2.7-fold). RhIL-6 had no effect on alpha 2-macroglobulin, whereas fibronectin, albumin and transferrin decreased to 64, 56 and 55% of controls. In the cases of serum amyloid A, haptoglobin, alpha 1-antichymotrypsin, alpha 1-antitrypsin and alpha 1-acid glycoprotein, dexamethasone enhanced the action of rhIL-6. We conclude that rhIL-6 controls the acute phase response in human liver cells.

The effect of age on several messenger RNAs coding for non-specific immune factors were determined in liver RNA isolated from 6-, 12-, 24-, 29- and 37-month-old male Fischer Rats. The levels of complement factors C3 and C4, complement protein factor B, and alpha 1-acid glycoprotein were determined by dot blot hybridization using cDNA probes. All four mRNAs increased slightly between 6 and 29 months of age. Only the mRNAs coding for complement factors C3 and C4 decreased significantly after 29 months of age. In addition, Northern blot analysis of the RNA preparations showed that the size of the four mRNA species did not change with increasing age. There was no evidence for age-related changes in the post-transcriptional processing or degradation of the mRNA species coding for complement factors C3 and C4, complement protein factor B, and alpha 1-acid glycoprotein.

To study the effect of intraabdominal sepsis on hepatic protein synthesis, male Sprague-Dawley rats underwent celiotomy with either cecal ligation and puncture (CLP) or sham operation. Eight and sixteen hours later total hepatic protein synthesis was measured by flooding dose technique. Specific synthetic rates of structural or secreted hepatic proteins were further studied 16 hr after CLP in an isolated perfused liver model. Total hepatic protein synthesis was significantly elevated at 16 hr (59 +/- 6%/day vs 37 +/- 6%/day, P less than 0.05), but not 8 hr post-CLP. Structural hepatic protein synthesis was unchanged after CLP; however, the synthetic rates of the acute-phase secretory proteins alpha 1-acid glycoprotein, transferrin and complement component C3 were significantly increased 16 hr after CLP. However, the albumin synthetic rate was not increased during sepsis. We conclude that sepsis causes augmentation of hepatic protein synthesis primarily to increase acute-phase proteins for host defense.

Expression of hepatocyte protein gene-products changes as cells adapt to the environment of tissue culture. We examined such changes, using "giant" two-dimensional (2-D) gel electrophoresis. Over 80 cellular and secreted proteins, about 3% of the 2500 we examined, were found to change spontaneously during the 20-h culture period. The magnitude of these changes was estimated to range from 1.5- to nearly 100-fold. If dexamethasone is included in the culture medium, the overall production of secreted proteins is maintained, but in addition it exerts major influences on the expression of individual protein gene-products. Ten of the spontaneous changes are enhanced and eight are unaffected, but most are substantially retarded (32) or even reversed (27). There are also large inductions or repressions in 12 proteins that do not change spontaneously in culture. We have tentatively identified tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, gamma-fibrinogen, and alpha 1-acid glycoprotein to be among those induced by dexamethasone. Evidently, many more changes occur as these cells adapt to tissue culture than was previously appreciated. These results emphasize the power of 2-D gel analysis in monitoring the protein phenotype of cells. We have also shown that glucocorticoids are important in maintaining the overall synthesis of secreted proteins and the protein phenotype of isolated hepatocytes in primary culture.

By measuring the incorporation of 14C-DL-leucine a decreased capacity of isolated perfused steptozotocin diabetic rat liver to synthetize plasma total proteins, albumin and the seromucoid fraction was found as compared with a control. The relative rate of synthesis of the seromucoid proteins calculated as proportional to the rate of synthesis of albumin or the total plasma proteins was approximately twice as great as in control liver. 14C-DL-leucine was also incorporated in perfused liver into the nonprotein but non-dialysable sugar-peptide fraction but the synthesis of the glycopeptide components of this fraction was markedly reduced in diabetic rat liver.

Serum and liver zinc concentration, amino acid uptake by liver, seromucoid content, and alpha2-macrofetoprotein production were measured in vaccinated as well as nonimmune rats exposed to either virulent (SCHU S4) or attenuated (LVS) strains of Francisella tularensi. It appears that liver damage (pyogranulomatous lesions) must occur before there is any alteration in the above variables. The presence of bacteria in the liver is not of itself sufficient to lead to the onset of systemic, induced metabolic sequelae (IMS). The occurrence of zinc redistribution in all instances of increased serum protein synthesis may imply a necessary relationship between these two sequelae. Amino acid redistribution does not appear to be linked to serum protein synthesis. An endogenous mediator of systemic IMS can be detected in tularemic rats by injection of the serum of these animals into healthy recipients. The occurrence of zinc redistribution and increased serum protein synthesis in some groups of rats in the absence of amino acids uptake by liver, as well as the apparent differential dose responsiveness of these responses, are suggestive of a multiplicity of endogenous mediators.

Two different forms of alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein were found in primary cultures of rat hepatocytes. After a 2.5-h labeling period with [35S]methionine the high-mannose-type precursor of alpha 1-proteinase inhibitor (Mr 49000) and alpha 1-acid glycoprotein (Mr 39 000) and the mature-complex-type alpha 1-proteinase inhibitor (Mr 54 000) and alpha 1-acid glycoprotein (Mr 43 000-60 000) could be immunoprecipitated from the cells, but only the complex-type forms of the two glycoproteins were secreted into the hepatocyte media. When hepatocytes were incubated with the mannosidase I inhibitor 1-deoxymannojirimycin at a concentration of 4 mM, the 49 000-Mr form of alpha 1-proteinase inhibitor and the 39 000-Mr form of alpha 1-acid glycoprotein could be detected in the cells as well as in their media. Neither the secretion of alpha 1-proteinase inhibitor nor that of alpha 1-acid glycoprotein was impaired by 1-deoxymannojirimycin. While alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, secreted by control cells, were resistant to endoglucosaminidase H, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, secreted by hepatocytes treated with 4 mM 1-deoxymannojirimycin, could be deglycosylated by endoglucosaminidase H. When the [3H]mannose-labeled oligosaccharides of alpha 1-proteinase inhibitor, secreted by 1-deoxymannojirimycin-treated hepatocytes, were cleaved off by endoglucosaminidase H and analyzed by Bio-Gel P-4 chromatography, they eluted at the position of Man9GlcNAc, indicating that mannosidase I had been efficiently inhibited. 1-Deoxymannojirimycin did not inhibit the synthesis or the cotranslational N-glycosylation of alpha 1-proteinase inhibitor or alpha 1-acid glycoprotein.

Automated Edman degradation of alpha 1-acid glycoprotein from serum (serum AGP) was successful only after the desialylated, reduced, and alkylated protein had been treated with pyroglutamate aminopeptidase. The sequence is (less than Glu)-Asn-Pro-Glu-Pro-Ala-X-Ile-Thr-Leu-Gly-Ile-Pro-Ile-. For radioactive sequencing of the NH2 terminus of the intracellular precursor form (liver AGP) of serum AGP, protein was labeled by incubating liver cells in suspension with either [3H]proline or [3H]isoleucine for a 15-min period. Liver AGP was separated from serum AGP by ion exchange chromatography. Between 78 and 90% of the radioactivity incorporated into total AGP was associated with liver AGP, indicating that little conversion of newly synthesized liver AGP to serum AGP had occurred during the 15-min incubation period. Liver AGP was then isolated by immunoprecipitation. The antigen-antibody complex was exposed to 15 Edman degradation cycles; however, no radioactivity was released. Automated Edman degradation of liver AGP which had been extracted from the immunoprecipitate with dilute acid and subsequently treated with pyroglutamate aminopeptidase yielded a sequence of proline and isoleucine identical with that in serum AGP. We conclude that processing of the intracellular precursor form of AGP does not involve a proteolytic trimming near the NH2 terminus.

The effect of the alpha-glucosidase inhibitor N-hydroxyethyl-1-deoxynojirimycin (Bay m 1099) on the glycosylation and secretion of alpha 1-antitrypsin (three complex type oligosaccharide chains) and of alpha 1-acid glycoprotein (six complex type oligosaccharide chains) was studied in rat hepatocyte primary cultures. In the presence of 4 mM Bay m 1099 the processing of high-mannose to complex type oligosaccharides was partially inhibited leading to the secretion of alpha 1-antitrypsin and alpha 1-acid glycoprotein carrying a mixture of both high-mannose and complex type oligosaccharides. The major part of alpha 1-antitrypsin secreted by Bay m 1099 treated cells still carried two complex type oligosaccharide chains, the majority of alpha 1-acid glycoprotein carried three to five. Despite its effects on protein glycosylation Bay m 1099 did not lead to pronounced changes in the synthesis or secretion of alpha 1-antitrypsin, alpha 1-acid glycoprotein or albumin. At concentrations of Bay m 1099 lower than 0.5 mM no inhibitory effect on oligosaccharide trimming could be observed. After removal of Bay m 1099 from hepatocytes its inhibitory effect on protein glycosylation was immediately reversible.

We measured the serum concentration of alpha 1-acid glycoprotein (alpha 1-AGP) and we evaluated the content of its hepatic mRNA in rats after 17 alpha-ethynyloestradiol treatment or after turpentine-induced acute inflammation, or after both treatments performed simultaneously. We have also studied the affinity of serum alpha 1-AGP for concanavalin A under these conditions. Both types of stimuli induce a marked retention of the glycoprotein on free concanavalin A. The serum concentration of alpha 1-AGP is increased about 14-fold compared with that in control rats when a single pharmacological dose (50 micrograms) or multiple injections of 17 alpha-ethynyloestradiol are administered. This increase is greater in turpentine-oil-injected rats (about 21-fold) and reaches a maximum (about 32-fold) in rats injected with 17 alpha-ethynyloestradiol plus turpentine oil; this increase in alpha 1-AGP corresponds to the addition of the effects of the two inducing agents. Similar changes are also observed either in the alpha 1-AGP mRNA content as estimated by using an alpha 1-AGP-specific cDNA probe, or in the amount of translatable alpha 1-AGP mRNA. The results indicate that: after a high dose of 17 alpha-ethynyloestradiol and after acute inflammation, the increase of the alpha 1-AGP serum concentration is due to an accumulation of the alpha 1-AGP mRNA; different mechanisms and/or pathways are probably involved in regulating the synthesis of alpha 1-AGP under various stimuli; 17 alpha-ethynyloestradiol as well as acute inflammation seem to control the glycosylation process of alpha 1-AGP in an identical manner.

The effect of dexamethasone on the synthesis of acute phase proteins has been studied in primary cultures of rat hepatocytes. In the absence of dexamethasone no detectable amounts of alpha 2-macroglobulin were synthesized by hepatocytes cultured for 1 day. alpha 2-Macroglobulin synthesis was induced by dexamethasone concentrations of 10(-8) M or higher with a maximum at a concentration of 10(-7) M. alpha 1-Acid glycoprotein was synthesized in the absence of dexamethasone; however, its synthesis was also greatly stimulated by dexamethasone concentrations of 10(-8)-10(-6) M. Synthesis of alpha 1-proteinase inhibitor was stimulated only 1.4-fold at a dexamethasone concentration of 10(-7) M. The kinetics of induction of alpha 2-macroglobulin and alpha 1-acid glycoprotein were studied at a dexamethasone concentration of 10(-7) M. After an initial lag phase of 3 h the synthesis of both proteins showed a steady increase during 2 days. Synthesis of albumin remained unchanged under these experimental conditions. Unlike alpha 2-macroglobulin and alpha 1-acid glycoprotein tyrosine aminotransferase activity increased already during the first 3 h of induction by dexamethasone with a maximum at 12 h followed by a slight decrease.

We used a system of co-culture of adult rat hepatocytes with another epithelial cell type from rat liver to study the synthesis of two acute-phase reactants, alpha-1 acid glycoprotein (alpha 1AGP) and the third component of complement (C3), and we have obtained long-term secretion of these two proteins. After a period of adaptation corresponding to the first 2-4 days of the co-culture, hepatocytes secreted C3 and alpha 1AGP for at least 2 weeks at a mean level higher than that observed in the first days of a pure culture of hepatocytes. When pulse-chase analysis was performed on day 6 of co-culture, kinetics of synthesis of alpha 1AGP and C3 were the same as those observed on day 1 of a conventional culture of pure hepatocytes. Furthermore, intracellular and extracellular alpha 1AGP had Mr values respectively of 39,000 and of 42,000-52,000, identical with those observed in pure cultures of hepatocytes. Similarly, the molecular size and subunit structures of C3 were the same in co-culture and in cultures, indicating an identical processing of this protein. C3 produced in co-culture was also haemolytically active. Therefore, the system of adult hepatocytes co-cultured with this liver epithelial cell provides a physiological system in vitro which permits long-term synthesis of the two acute-phase reactants C3 and alpha 1AGP. This model opens the possibility to study the modulation of the synthesis of these two proteins during a long period by inflammatory agents or by hormones.

The hepatic synthesis of four "acute phase reactants" (APR), i.e., fibrinogen, alpha 1-acid glycoprotein, alpha 2-macroglobulin, and haptoglobin has been investigated in rats suffering from turpentine-induced inflammation. To follow the change in the rates of synthesis of the four APR, their concentrations were measured by immunonephelemetry in both the plasma and the hepatic microsomal fraction at various times after injury. A synchronous increase in the concentrations of these four proteins was observed in the liver (maximum 24 hours) as well as in the plasma (maximum 40 hours) of the same animals. In parallel, the site of their synthesis was localized in liver sections by light and electron microscopy using direct immunoperoxidase labeling. Of the liver cells, only the hepatocytes were labeled. In the early period of the inflammatory reaction (10 to 16 hours), synthesizing cells were detected principally in the periportal zone, but later (24 hours), the labeled area was extended to nearly the entire hepatic lobule. When serial sections of liver were examined at that time, the same cells were found to contain simultaneously the four APR. Within the cells examined by electron microscopy, the four proteins were localized in the secretory pathway, i.e., rough and smooth endoplasmic reticulum, Golgi apparatus, and secretory vacuoles. Therefore, we conclude that: (1) the experimental inflammatory reaction induces a synchronous increase in the synthesis of four APR by the liver; (2) this increased synthesis apparently results from an increased number of synthesizing hepatocytes; (3) these cells have a preferential periportal localization; and (4) individual hepatocytes are not specialized in the synthesis of a single plasma protein.

Albumin, fibrinogen, alpha 1-acid glycoprotein and cysteine proteinase inhibitor were determined by electroimmunoassay in the media of primary cultures of rat hepatocytes exposed to dialysed supernatants of rat, mouse and human macrophages or to recombinant human and murine interleukin 1 and tumour necrosis factor. Recombinant cytokines in the range of 1 to 1000 ng/ml caused only reduction of albumin synthesis and slight stimulation of alpha 1 acid glycoprotein production while crude preparations of macrophage cytokines elicited typical acute phase response. The results suggest that interleukin 1 or tumour necrosis factor are not likely the principal mediators responsible for the direct stimulation of normal rat hepatocytes to acute phase protein synthesis.

The threonine analog beta-hydroxynorvaline (Hnv) is an inhibitor of asparagine-linked glycosylation. In the presence of the analog hepatocytes synthesized immunoreactive alpha 1-acid glycoprotein with 0-6 oligosaccharide chains. Pulse-chase experiments were conducted to compare the rates of secretion of alpha 1-acid glycoprotein from untreated, tunicamycin-treated, and Hnv-treated cells. Partially glycosylated (1-5 oligosaccharide chains) and unglycosylated (tunicamycin-inhibited) molecules exited the cells more slowly than native alpha 1-acid glycoprotein. In addition, secretion of fully glycosylated (6 oligosaccharide chains) alpha 1-acid glycoprotein was retarded in Hnv-treated cells when compared to controls. The slowest rate of secretion was exhibited by the unglycosylated form from Hnv-treated cells. These results suggest that Hnv-induced changes either in the extent of glycosylation or in the peptide sequence of alpha 1-acid glycoprotein can interfere with its transport through the cell. The major intracellular forms of alpha 1-acid glycoprotein from control and Hnv-treated cells were endoglycosidase H-sensitive and contained Man9-8 GlcNAc2 oligosaccharide structures. The oligosaccharide chains on the secreted molecules from control and Hnv-treated cells were entirely of the endoglycosidase H-resistant, complex type.

The isolated rat liver perfused for 12 hours at pH 7.10 with a suspension of bovine erythrocytes in Krebs-Ringer bicarbonate buffer containing 3 per cent bovine serum albumin has been used as a test system to study effects of glucagon and of dexamethasone in the presence and absence of insulin on net biosynthesis of rat serum albumin, fibrinogen, alpah1-acid glycoprotein, alpha2-(acute phase) globulin, and haptoglobin. Quantitative measurement of perfusate glucose, amino acid nitrogen, and urea affords a basis for determining net glucose and nitrogen balance in the perfusion system. Although the dose of dexamethasone (total 1.0 mug.) used was insufficient to induce synthesis of alpha2-acute phase globulin, net syntheses of albumin, fibrogen, alpha1-acid glycoprotein, and haptoglobin were increased. Glucagon given with dexamethasone depressed albumin and haptoglobin synthesis markedly, but not that of fibrinogen and alpha1-acid glycoprotein. Glucagon with dexamethasone markedly enhanced ureogenesis and glycogenolysis and elicited an exaggerated negative nitrogen balance. The unfavorable effects of glucagon on albumin and haptoglobin synthesis and on nitrogen balance were reversed by giving insulin simultaneously. It is emphasized that insulin is essential for positive nitrogen balance.

Primary mouse hepatocytes were treated with the acute-phase mediators interleukin-1, interleukin-6, and glucocorticoids, singularly and in combination, in order to delineate the spectrum of proteins induced by the stimulatory factors. As found for rat and human liver cells, mouse hepatocytes responded to the cytokines by increasing production of acute-phase proteins, which in mice include haptoglobin, alpha 1-acid glycoprotein, complement C-3, serum amyloid A, and hemopexin. Serum amyloid A was unusual in that only the acidic peptide form responded to treatment with IL-1 and IL-6; the more basic form remained unchanged. In addition, an unidentified secretory protein was induced only by mixtures containing IL-6. The present study shows that a combination of IL-1, IL-6, and glucocorticoids is required for regulation of acute-phase plasma protein production in mouse liver cells.

Hybridization studies using specific cDNA probes have been used to determine the specific mRNA levels for apolipoproteins B and E, alpha 1 acid glycoprotein and beta actin in extracts of rat liver. Injection of rats with recombinant mouse tumor necrosis factor had led to a rapid increase in liver mRNA levels for alpha 1 acid glycoprotein (x 12) and for beta actin (x 2.5) whereas mRNA levels for Apolipoprotein B and E remained stable over the same period.

Human squamous carcinoma (COLO-16) cells release factors which specifically stimulate the synthesis of major acute-phase plasma proteins in human and rodent hepatic cells. Anion exchange, hydroxyapatite, lectin, and gel chromatography of conditioned medium of COLO-16 cells result in separation into three distinct forms of hepatocyte-stimulating factors (designated HSF-I, HSF-II, and HSF-III) with apparent molecular weights of 30,000, 50,000 and 70,000, respectively. None of the preparations contains detectable amounts of thymocyte-stimulating activity. Each of the three HSF forms stimulates the accumulation of mRNA for alpha 1-antichymotrypsin in the human hepatoma cell line, HepG2. When the same factors were added to primary cultures of adult rat hepatocytes, the expression of the same set of plasma proteins was modulated as by nonfractionated medium. The hormonally induced accumulation of mRNA for acute phase proteins is qualitatively comparable to that occurring in the liver of inflamed rats. Unlike in human cells, in rat liver cells dexamethasone acts additively and synergistically with HSFs. The only functional difference between the three HSF forms lies in the level of maximal stimulation. HSF-I represents the predominant form produced by normal human keratinocytes and closely resembles in molecular size and isoelectric point the activity produced by activated peripheral blood monocytes while the larger molecular weight forms are more prevalent in human as well as mouse squamous carcinoma cells. The observation that HSFs from different sources elicit essentially the same pleiotropic response in hepatic cells led to the hypothesis that the species-specific reaction of adult liver cells to inflammatory stimuli is pre-programmed and that the function of any HSF is to trigger and tune the execution of this fixed cellular process.

Ultrasonic extracts of rough and smooth endoplasmic reticulum fraction and Golgi fractions from rat liver were examined by immunoelectrophoresis using antiserum to alpha 1-acid glycoprotein. Rough endoplasmic reticulum fractions contained only sialic acid free alpha 1-acid glycoprotein, whereas smooth endoplasmic reticulum and Golgi fractions also contained sialic acid containing alpha 1-acid glycoprotein. Determination of the sialic acid contents of immune precipitates isolated from the extracts suggested that the Golgi complex was the main site of addition of sialic acid to alpha 1-acid glycoprotein. Immunological studies on puromycin extracts of polyribosomes showed that polypeptide chains of alpha 1-acid glycoprotein and albumin were assemble mainly on membrane-bound polyribosomes. Evidence is presented from incorporation studies with labelled leucine and glucosamine that initial glycosylation of alpha 1-acid glycoprotein occurs mainly or entirely after release of nascent polypeptide from the ribosomal site.