Elevated plasma concentrations of haptoglobin in European brown bears during hibernation

Haptoglobin (Hp), a hemoglobin-binding protein, is known as an acute phase protein and increases during the acute phase of inflammation in most mammals. We reported previously in brown bears that the mean Hp concentrations were higher in blood samples obtained in winter than those in spring. To examine a possible relation of the seasonal variations of Hp to hibernation, in the present study, we measured the plasma concentrations of Hp as well as some other acute phase proteins (alpha(2)-macroglobulin, alpha(1)-antitrypsin, C-reactive protein) in 6 European brown bears (Ursus arctos), from which blood samples were obtained at 5-6 different months of year including February, the time of hibernation. The Hp concentrations showed clear seasonal variations, being highest in February. The alpha(2)-macroglobulin concentrations also showed a similar but much smaller rise in February, but those of alpha(1)-antitrypsin and C-reactive protein did not show any seasonal variations. Our results suggest that the seasonal variation of plasma Hp concentration in brown bears is associated with a hibernation-specific mechanism more than that of acute phase response.     

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.     

IL-6 modulates the synthesis of a specific set of acute phase plasma proteins in vivo

Human rIL-6, produced either in COS cells or Escherichia coli, similarly stimulates the production of acute phase plasma proteins in cultured human and rat hepatoma cells. This anabolic effect in hepatoma cells suggested a potential in vivo role of the cytokine in mediating the hepatic response to inflammation. Injection of IL-6 into adult male rats elicited a cytokine-specific change in the liver expression of acute phase proteins. As predicted from in vitro studies, glucocorticoids were needed to achieve a maximal IL-6 response in vivo. Optimal conditions were found to be two i.p. injections of 35 to 120 micrograms IL-6 and 65 micrograms dexamethasone per kg body weight administered at 12-h intervals. Within 24 h, the plasma concentrations for alpha 2-macroglobulin, fibrinogen, thiostatin, and hemopexin were increased to levels approximating those observed in acute phase animals. These results support the notion that direct interaction of IL-6 with the liver is an essential part in initiating the hepatic acute phase reaction.     

Effect of endotoxin on plasma albumin and fibrinogen synthesis rates in rabbits as measured by the [14C]carbonate method

1. Rates of synthesis of plasma albumin and fibrinogen were measured by the [(14)C]carbonate method in normal rabbits and in animals that received a single intravenous injection of Shigella endotoxin 14-48hr. earlier. 2. The accuracy of the method was improved by introducing refinements into procedures for measuring (14)C radioactivities associated with both urea and proteins that are lost from the plasma during the synthesis interval. 3. The synthesis interval (time between injecting carbonate and measuring specific radioactivities of protein guanidine carbon in plasma) can be shortened with advantage to 3-4hr. 4. Injection of endotoxin markedly decreased the fractional rate of loss in the first few hours of injected radioiodine-labelled fibrinogen and to a smaller extent of similarly labelled albumin from the plasma. The absolute rate of synthesis of fibrinogen was increased in endotoxin-treated rabbits by more than 400% compared with normal animals, and the rate of synthesis of albumin was increased by about 60%.     

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.     

Regulation of rabbit acute phase protein biosynthesis by monokines.

We defined the acute phase behaviour of a number of rabbit plasma proteins in studies (in vivo) and studied the effects of monokine preparations on their synthesis by rabbit primary hepatocyte cultures. Following turpentine injection, increased serum levels of C-reactive protein, serum amyloid A protein, haptoglobin, ceruloplasmin, and decreased concentrations of albumin were observed. In contrast to what is observed in man, concentrations of alpha 2-macroglobulin and transferrin were increased. Co-culture of primary hepatocyte cultures with lipopolysaccharide-activated human peripheral blood monocytes or incubation with conditioned medium prepared from lipopolysaccharide-activated human or rabbit monocytes resulted in dose-dependent induction of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and depression of albumin synthesis, while C-reactive protein synthesis and mRNA levels remained unchanged. A variety of interleukin-1 preparations induced dose-dependent increases in the synthesis and secretion of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and decreased albumin synthesis. Human recombinant tumour necrosis factor (cachectin) induced a dose-dependent increase in synthesis of haptoglobin and ceruloplasmin. In general, human interleukin-1 was more potent than mouse interleukin-1 and tumour necrosis factor. None of the monokines we studied had an effect on C-reactive protein synthesis or mRNA levels. These data confirm that C-reactive protein, serum amyloid A, haptoglobin and ceruloplasmin display acute phase behaviour in the rabbit, and demonstrate that, in contrast to their behaviour in man, alpha 2M and transferrin are positive acute phase proteins in this species. While both interleukin-1 and tumour necrosis factor regulate biosynthesis of a number of these acute phase proteins in rabbit primary hepatocyte cultures, neither of these monokines induced C-reactive protein synthesis. Comparison of these findings with those in human hepatoma cell lines, in which interleukin-1 does not induce serum amyloid A synthesis, suggests that the effect of interleukin-1 on serum amyloid A synthesis may be indirect.     

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.     

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.     

Binding of alpha 2-macroglobulin and haptoglobin to Actinomyces pyogenes

All 25 cultures of Actinomyces pyogenes tested in the present study bound 125I-labelled human alpha 2-macroglobulin with a mean binding of 65.6%. Thirteen cultures also bound 125I-labelled human haptoglobin with a mean of 51.5%. None interacted with fibrinogen, fibronectin, immunoglobulin G, or albumin. Twenty-eight cultures representing other species of actinomycetaceae did not show any interaction with alpha 2-macroglobulin, haptoglobin, and other plasma proteins tested. The binding of alpha 2-macroglobulin and haptoglobin to A. pyogenes was saturable and could be completely inhibited by the respective unlabelled plasma proteins. The binding of alpha 2-macroglobulin could not be inhibited by unlabelled haptoglobin. On the other hand, alpha 2-macroglobulin blocked the binding of haptoglobin, possibly by steric hindrance. Treatment of the bacteria with trypsin reduced their binding activities for alpha 2-macroglobulin and haptoglobin indicating the protein nature of the binding sites. Exposure to heat (1 h, 80 degrees C) significantly diminished the binding activity for haptoglobin, but not that for alpha 2-macroglobulin. The binding of alpha 2-macroglobulin and haptoglobin could be an important feature in the classification of A. pyogenes among the members of actinomycetaceae.     

Sequence and acute phase regulation of rat alpha 1-inhibitor III messenger RNA

cDNA clones coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from an acute phase rat liver library. The isolates could be divided into four groups with characteristic BamHI restriction fragment patterns. The identity of the prototype clone pRLA1I3/2J was established by comparison with the published amino acid sequence of the purified protein. It codes for a 1477-amino acid precursor polypeptide with a 24-residue signal peptide. The mature protein shares 58% overall sequence identity with rat alpha 2-macroglobulin and contains a typical internal thiolester sequence. Twenty-two of its twenty-three cysteinyl residues are conserved with alpha 2-macroglobulin implying similar tertiary structure. However, the prototype alpha 1-inhibitor III sequence differed significantly from the rat and human alpha 2-macroglobulin sequences in its bait region suggesting alpha 1-inhibitor III possesses proteinase inhibitory specificities different from those of alpha 2-macroglobulin. The variant alpha 1-inhibitor III clone pRLA1I3/2J from a second cDNA group also differed from the prototype in the bait region coding sequence, although both specify similar signal peptides and NH2 termini. The observation of variant cDNA classes suggests that acute phase rat livers produce a heterogeneous mixture of alpha 1-inhibitor III mRNA molecules. Evidence was obtained for the presence of at least four different alpha 1-inhibitor III-related genes in the rat genome. During the first 24 h of an acute phase response the abundance of hepatic alpha 1-inhibitor III mRNA was decreased 3-4-fold. This decrease was of the same order of magnitude as the reported reduction of the corresponding plasma protein concentration, suggesting that in the early phase of the acute inflammatory response the plasma concentration of this protein is mainly controlled through the abundance of its hepatic mRNA.     

A stable long-term hepatocyte culture system for studies of physiologic processes: cytokine stimulation of the acute phase response in rat and human hepatocytes.

Prior studies on the in vitro hepatic acute phase response have involved either hepatoma cell lines or conventional short-term cultures of primary hepatocytes. No data are available on the response of primary hepatocytes in stable long-term culture systems. In this study, the acute phase response of rat and human hepatocytes in a new long-term culture system was examined in response to interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), and tumor necrosis factor alpha (TNF-alpha). The cultured cells were sandwiched between two layers of collagen in a (double-gel) configuration which has been shown to preserve both hepatocyte function and morphology over prolonged periods of time. The stability of this culture configuration enabled us to investigate, for the first time, the temporal aspects of the response in addition to the effects of the mediators on protein secretion. Exposure of rat hepatocytes to IL-6 after culture for 16 days resulted in a 2-fold reduction of albumin secretion and a 15-fold increase in the secretion rates of fibrinogen and alpha 2-macroglobulin. In all instances, the peak response occurred at 48 h after IL-6 exposure, and all protein secretion rates returned to pretreatment values within 5 days posttreatment. Changes in the mRNA levels of these proteins in response to IL-6 corresponded with those changes seen with the secreted products, indicating pretranslational regulation. Administration of IL-1 beta to rat hepatocyte produced a similar decline of albumin secretion and a 5-fold increase of fibrinogen secretion, whereas alpha 2-macroglobulin secretion remained undisturbed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteins in cerebrospinal fluid and plasma of the pouch young tammar wallaby (Macropus eugenii) during development

The concentrations of total protein and albumin in cerebrospinal fluid (CSF) and plasma of tammar wallaby pouch young (Macropus eugenii) from birth until leaving the pouch have been measured. Total protein in CSF increased from birth (about 240 mg/100 ml) to 15-20 days postnatal (about 400 mg/100 ml) after which it declined. Albumin showed a proportionately greater increase from around 40 mg/100 ml to over 130 mg/100 ml, followed by decline after 75 days. Total protein and albumin in plasma increased throughout the period studied. Other proteins identified in CSF and plasma were: fetuin, alpha 2-macroglobulin, transferrin, alpha-lipoprotein, beta-lipoprotein, immunoglobin G and fibrinogen. One protein was only present in early pouch young (up to about 40 days) and was presumed to be the tammar equivalent of alpha-fetoprotein.     

Twelve serum protein profiles in children with acute nonbacterial gastroenterocolitis.

In thirty children hospitalized with acute benign, short-duration gastroenterocolitis, no obligate pathogens were isolated from stools. Five bleedings were established from each patient in order to obtain the protein profiles of albumin, orosomucoid, haptoglobin, alpha2-macroglobulin, ceruloplasmin, transferrin, C3-component, C-reactive protein, immunglobulins IgG, IgA, IgM and IgD. The proteins were quantitated by the single radial immunodiffusion method. The initial drop in some of the proteins followed may be related to general protein loss, negative nitrogen balance or hemodilution. The absence of a significant increase in all the investigated immunoglobulin classes contrasted with remarkable increase in haptoglobin and orosomucoid, both reaching normal levels in late convalescence. C-reactive protein could be demonstrated in half of the children showing early normalization with disappearance of clinical symptoms. In contrast to ceruloplasmin and C3- component, alpha2-macroglobulin was not involved in the acute phase protein reaction.     

Effect of leukocytic endogenous mediator on C-reactive protein in rabbits.

Elevation in the plasma levels of the acute phase proteins--C-reactive protein (C-RP) and fibrinogen--were found after injection of leukocytic endogenous mediator (LEM) into rabbits. C-RP in the plasma was elevated 8 hr after injection of LEM, and maximum elevation occurred 24 hr after injection. Injection of LEM into rabbits also produced alterations in body temperature, in levels of plasma iron and zinc, and in the number of neutrophils in the peripheral blood.     

Plasma levels of immunoreactive ceruloplasmin and other acute phase proteins during lactation

Lactation elevates plasma copper as well as oxidase activity levels of the copper-containing, acute phase protein ceruloplasmin (Cp). The present study provides an initial inquiry into the mechanisms behind these changes. Plasma obtained from 12 lactating women, 1 month postpartum, displayed a greater percentage increase in immunoreactive Cp levels (mean increase = 89%) than in plasma copper (mean = 66%) or Cp oxidase activity (mean = 42%). Lactation did not increase plasma content of C-reactive protein or alpha 1-antitrypsin but significantly elevated haptoglobin concentrations. Plasma alpha 2-macroglobulin contents correlated with immunoreactive Cp levels in lactating women but not in controls. These results strengthen the hypothesis that plasma content of individual acute phase proteins is regulated by both overlapping and individualized processes. In addition, the present findings raise the possibility that lactation increases both Cp synthesis and plasma turnover time of Cp-bound copper.     

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.     

Inflammatory response following acute magnesium deficiency in the rat

The importance of inflammatory processes in the pathology of Mg deficiency has been recently reconsidered but the sequence of events leading to the inflammatory response remains unclear. Thus, the purpose of the present study was to characterize more precisely the acute phase response following Mg deficiency in the rat. Weaning male Wistar rats were pair-fed either a Mg-deficient or a control diet for either 4 or 8 days. The characteristic allergy-like crisis of Mg-deficient rats was accompanied by a blood leukocyte response and changes in leukocytes subpopulations. A significant increase in interleukin-6 (IL-6) plasma level was observed in Mg-deficient rats compared to rats fed a control diet. The inflammatory process was accompanied by an increase in plasma levels of acute phase proteins. The concentrations of alpha2-macroglobulin and alpha1-acid glycoprotein in the plasma of Mg-deficient rats were higher than in control rats. This was accompanied in the liver by an increase in the level of mRNA coding for these proteins. Moreover, Mg-deficient rats showed a significant increase in plasma fibrinogen and a significant decrease in albumin concentrations. Macrophages found in greater number in the peritoneal cavity of Mg-deficient rats were activated endogenously and appeared to be primed for superoxide production following phorbol myristate acetate stimulation. A high plasma level of IL-6 could be detected as early as day 4 for the Mg-deficient diet. Substance P does not appear to be the initiator of inflammation since IL-6 increase was observed without plasma elevation of this neuropeptide. The fact that the inflammatory response was an early consequence of Mg deficiency suggests that reduced extracellular Mg might be responsible for the activated state of immune cells.