The association of acute phase protein genes with the nuclear matrix of rat liver during experimental inflammation

The association of acute phase protein genes with the nuclear matrix in livers from healthy rats and rats suffering from inflammation was studied. alpha 1-Acid glycoprotein and transthyretin are synthesized at low levels in normal liver and no matrix association of their genes was observed. Albumin, transferrin and the beta-chain of fibrinogen are synthesized at much higher levels in normal liver and their genes were found to be associated with the nuclear matrix. An inflammation induced increase in synthesis of alpha 1-acid glycoprotein and the beta-chain of fibrinogen resulted in stronger matrix association of their genes. However, inflammation induced decrease in the synthesis of albumin did not influence matrix association of its gene.     

Rat choroid plexus specializes in the synthesis and the secretion of transthyretin (prealbumin). Regulation of transthyretin synthesis in choroid plexus is independent from that in liver

Synthesis of total protein and of transthyretin in rat choroid plexus was studied by measuring the incorporation of radioactive leucine into proteins in choroid plexus tissue incubated in vitro. About 20% of the protein newly synthesized in choroid plexus and about 50% of the newly synthesized protein secreted into the medium was transthyretin. Evidently, the choroid plexus is very active in the biosynthesis of this carrier protein for thyroid hormones and could be an important link in the chemical communication between the body and the central nervous system. Acute inflammation, which leads to a profound rearrangement of the pattern of plasma protein synthesis rates in the liver, produced distinct changes in the levels for plasma protein mRNAs in the liver. The levels of the mRNAs for alpha 1-acid glycoprotein and major acute phase alpha 1-protein increased more than 30-fold, those for transthyretin and albumin decreased to 27 and 57% of normal, respectively. The pattern of the observed changes in the levels of mRNAs for plasma proteins in the liver was independent of whether the acute inflammation was produced by subcutaneous injection of turpentine or intraperitoneal injection of a suspension of talcum. However, levels of transthyretin mRNA in choroid plexus were affected only very slightly, or not at all. Apparently, transthyretin synthesis in liver and choroid plexus is regulated independently during the acute phase response. No mRNA was detected in choroid plexus for albumin, alpha 1-acid glycoprotein, and major acute phase alpha 1-protein under any conditions.     

Recombinant human interleukin-6 (IL-6/BSF-2/HSF) regulates the synthesis of acute phase proteins in human hepatocytes

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.     

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.     

High prealbumin and transferrin mRNA levels in the choroid plexus of rat brain

Expression of plasma protein genes in various parts of the rat brain was studied by hybridizing radioactive cDNA to RNA in cytoplasmic extracts. No mRNA could be detected in brain for the beta subunit of fibrinogen, major acute phase alpha 1-protein, alpha 1-acid glycoprotein and albumin. However, per g tissue, the choroid plexus contained at least 100 times larger amounts of prealbumin mRNA than the liver and about the same amount of transferrin mRNA as liver. No prealbumin mRNA was found in other areas of the brain. The results obtained suggest very active synthesis of prealbumin in choroid plexus, which would be an important link in the transport of thyroid hormones from the blood to the brain via the cerebrospinal fluid.     

Simplified elimination of rabbit anti-rat acute phase protein IgG that shows cross reactivity with albumin

Antibody preparations against rat acute phase proteins were tested for cross reactivity with other serum proteins, including rat albumin. Rabbit anti-rat a alpha1-acid glycoprotein and ceruloplasmin IgG purified on protein A-Sepharose did not show any cross reactivity with rat albumin, hemoglobin or transferrin. Rabbit anti-rat haptoglobin and -macroglobulin IgG purified on protein A-Sepharose showed a 39% and 30% cross reactivity with rat albumin and a 20% and 19% cross reactivity with rat hemoglobin. Because these proteins in whole serum were not adsorbed on Cibacron Blue F3-GA Sepharose, the albumin would be adsorbed on Cibacron Blue F3-GA Sepharose by the use of whole rat serum. Rabbit anti-rat haptoglobin and alpha2-macroglobulin IgG showing cross reactivity with albumin was simply eliminated.     

Messenger RNA activities of four acute phase proteins during inflammation

Poly(A)+ RNA isolated from the livers of normal rats and of rats suffering from an acute inflammation was translated in a cell-free translation system from rabbit reticulocytes. The translation products were immunoprecipitated with specific antisera against alpha 1-acid glycoprotein, alpha 2-macroglobulin, transferrin, alpha 1-proteinase inhibitor and albumin. 15 to 21 h after intramuscular injection of turpentine 73-, 66-, 2.8-, and 2-fold increases in translatable mRNAs for alpha 1-acid glycoprotein, alpha 2-macroglobulin, transferrin and alpha 1-proteinase inhibitor, respectively, were observed. For albumin a decrease in translatable mRNA to about 30% of controls was measured.     

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.     

Induction of acute phase proteins by dexamethasone in rat hepatocyte primary cultures

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.     

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.     

Acute-phase-response induction in rat hepatocytes co-cultured with rat liver epithelial cells.

The response of rat hepatocytes co-cultured with rat liver epithelial cells to conditioned medium (CM) from lipopolysaccharide (LPS)-activated monocytes was investigated by measuring the concentration of alpha 2-macroglobulin (alpha 2M), alpha 1-acid glycoprotein (AGP), albumin and transferrin, as well as the changes in glycosylation of alpha 1-acid glycoprotein. During an initial 8-day treatment with CM, concentrations of alpha 2M and AGP increased markedly over those of control culture, whereas concentrations of albumin and transferrin decreased. The glycosylation pattern of AGP indicated an important relative increase of the concanavalin A-strongly-reactive (SR) variant upon treatment. When CM addition to hepatocyte culture medium was stopped, the concentrations of the four proteins and the glycosylation pattern of AGP reverted to those of control cultures. Further addition (on day 15) to cultures of CM increased the concentration of alpha 2M and decreased albumin and transferrin concentrations. Although AGP concentrations did not increase above those of controls, the appearance of the SR variant was again stimulated by CM. These results show that, in co-culture, rat hepatocytes remain able to respond to repeated inflammatory stimuli.     

Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes

The three monokines interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interleukin-6 (IL-6) modulate acute phase plasma protein synthesis in adult human hepatocytes. Only IL-6 stimulates the synthesis of the full spectrum of acute phase proteins as seen in inflammatory states in humans, i.e. synthesis and secretion of C-reactive protein, serum amyloid A, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin and haptoglobin are increased while albumin, transferrin and fibronectin are decreased. IL-1 beta as well as TNF alpha, although having a moderate effect on the positive acute phase proteins and inhibiting the synthesis of fibrinogen, albumin and transferrin, fail to induce serum amyloid A and C-reactive protein. These data suggest that IL-6 plays the key role in the regulation of acute phase protein synthesis in human hepatocytes.     

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.     

The acute phase response to inflammation: the role of monokines in changes in liver glycoproteins and enzymes of glycoprotein metabolism

The role of monocyte derived factors in the acute phase response to inflammation is discussed. The kinetics of response of alpha 1-acid glycoprotein, sialyltransferase and albumin to a rat monokine preparation is described. There was an increase in synthesis of alpha 1-acid glycoprotein and sialyltransferase and a decrease in albumin synthesis following administration. However, the kinetics of response of sialyltransferase to the monokine was much slower than was found for the other two proteins. The possibility that sialyltransferase responds to a different monokine compared to the other acute phase proteins is discussed.     

Age-related changes in plasma proteins of analbuminemic rats

A mutant strain, Nagase analbuminemia rats (NAR), was established from Sprague-Dawley rats. Age-related changes in plasma proteins of NAR were investigated to obtain information of their abnormalities of protein metabolism. The total protein concentration in the serum of NAR of various ages was almost the same as that of normal rats of the same age. The albumin level of NAR was less than 0.05 mg/ml at all ages examined. The concentrations of serum alpha 1-antitrypsin, alpha-X protein, alpha 2-macroglobulin, transferrin, ceruloplasmin, IgG, IgA and IgM were higher in NAR than in normal rats except for the perinatal stage, but alpha 1-acid glycoprotein level in NAR was normal. The serum transferrin and ceruloplasmin levels were especially high in female adult NAR. The plasma fibrinogen concentration was also increased in NAR. These findings indicate that the normal total serum protein level of NAR was maintained by increase in the globulin concentration.     

Mining biomarkers in human sera using proteomic tools

One of the major difficulties in mining low abundance biomarkers from serum or plasma is due to the fact that a small number of proteins such as albumin, alpha2-macroglobulin, transferrin, and immunoglobulins, may represent as much as 80% of the total serum protein. The large quantity of these proteins makes it difficult to identify low abundance proteins in serum using traditional 2-dimensional electrophoresis. We recently used a combination of multidimensional liquid chromatography and gel electrophoresis coupled to matrix-assisted laser desorption/ionization-quadrupole-time of flight and Ion Trap liquid chromatography-tandem mass spectrometry to identify protein markers in sera of Alzheimer's disease (AD), insulin resistance/type-2 diabetes (IR/D2), and congestive heart failure (CHF) patients. We identified 8 proteins that exhibit higher levels in control sera and 36 proteins that exhibit higher levels in disease sera. For example, haptoglobin and hemoglobin are elevated in sera of AD, IR/D2, and CHF patients. The levels of several other proteins including fibrinogen and its fragments, alpha 2-macroglobulin, transthyretin, pro-platelet basic protein, protease inhibitors clade A and C, as well as proteins involved in the classical complement pathway such as complement C3, C4, and C1 inhibitor, were found to differ between IR/D2 and control sera. The sera levels of proteins, such as the 10 kDa subunit of vitronectin, alpha 1-acid glycoprotein, apolipoprotein B100, fragment of factor H, and histidine-rich glycoprotein were observed to be different between AD and controls. The differences observed in these biomarker candidates were confirmed by Western blot and the enzyme-linked immunosorbent assay. The biological meaning of the proteomic changes in the disease states and the potential use of these changes as diagnostic tools or for therapeutic intervention will be discussed.     

Modifications of serum glycoproteins the days following a prolonged physical exercise and the influence of physical training.

Eight male subjects (mean age 24.1 +/- 2.6 years) performed at intervals of 2 weeks successively a 3 h and two 2 h runs of different running speed. The days following the running there were moderate elevations of C-reactive protein, haptoglobin, alpha-1-acid glycoprotein, coeruloplasmin, transferrin, alpha-1-antitrypsin and plasminogen. There were small or no changes of albumin, alpha-2-macroglobulin and hemopexin. The elevations of the "acute phase reactants" were examined in three male subjects following a 2 h run before and after an endurance training period of 9 weeks. This demonstrated a decreased acute phase response after training as illustrated by the changes of C-reactive protein, haptoglobin and alpha-1-acid glycoprotein in spite of higher posttraining running speeds. Well-trained athletes have elevated levels of the serum protease inhibitors alpha-1-antitrypsin, alpha-2-macroglobulin and C1-inhibitor. These antiproteolytic glycoproteins might limit exercise-induced inflammatory reactions.     

Insulin modulation of acute-phase protein production in a human hepatoma cell line.

Insulin is widely used as a growth factor in hepatocyte culture but its effect on the production of acute-phase proteins has not been studied. By measuring four positive (fibrinogen, alpha 1-antitrypsin, alpha 1-acid glycoprotein, and alpha 1-antichymotrypsin) and four negative (albumin, prealbumin, transferrin, and retinol binding protein) acute-phase proteins produced by the Hep G2 hepatoma cell line, we have shown that insulin is an important modulator of acute-phase protein production. Our data show that insulin is able to inhibit the synthesis of prealbumin, transferrin, and fibrinogen. The results also show a complex interaction between insulin, interleukin 6, and glucocorticoids because insulin is able to inhibit the dexamethasone induction of alpha 1-antichymotrypsin, and in the presence of interleukin 6, dexamethasone is able to regulate the production of fibrinogen and prealbumin. The regulatory role of insulin in fibrinogen production was confirmed by pulse chase labeling followed by immunoprecipitation and fluorography.