Turpentine-induced Inflammation Research Articles

Translational regulation of ferritin synthesis in rat spleen: Effects of iron and inflammation

Translational control of ferritin synthesis was studied in rat spleen, and compared with that for liver, heart and brain, in response to iron and inflammation. Spleen concentrations of total RNA in the ribonucleoprotein (mRNP) fraction was comparable to that for liver, while polyribosomal RNA was less. Both fractions were ten-fold lower in heart and brain. In untreated animals, the mRNP fraction of all tissues had the largest portion of the ferritin mRNA, as determined by slot blot hybridization with 32P-labeled cDNA for the L subunit. Acute treatment with ferric ammonium citrate shifted the spleen ferritin mRNA to the polyribosome fraction. This was also so in liver but not in the heart and brain which took up much less iron. The findings were confirmed by hybridization studies of mRNPs and polyribosomes separated in sucrose gradients. Turpentine-induced inflammation also caused a shift in ferritin mRNA from the mRNP to the polyribosome fraction of spleen and liver, over 12 h. We conclude that as in liver, spleen ferritin synthesis is under translational control by iron, and that both tissues also respond to inflammation by shifting of ferritin mRNA to the polyribosomes.

Effect of turpentine-induced inflammation on the disposition kinetics of propranolol, metoprolol, and antipyrine in the rat.

Plasma concentrations after oral administration of the high extraction drug propranolol are increased in patients and animals with inflammation. This could be due to increased serum propranolol binding, but also to decreased first-pass metabolism. We studied the pharmacokinetics of 3 drugs in control rats and in rats with turpentine-induced inflammation: propranolol, which is bound extensively to alpha 1-acid glycoprotein (alpha 1-AGP); metoprolol, another high extraction drug, but which is negligibly bound to alpha 1-AGP; and antipyrine, a low extraction drug, not bound to serum proteins. After IV administration of propranolol in rats with inflammation, systemic clearance, volume of distribution, and free fraction decreased, and the area under the curve (AUC) increased, whereas the half-life did not change. As the systemic clearance of a high extraction drug such as propranolol depends on hepatic blood flow only, a fall in hepatic blood flow or transition to a low extraction situation should be postulated. After oral administration of propranolol, the AUC was increased 20-fold in rats with inflammation; as the decrease in free fraction was only 4-fold, it can be concluded that a considerable decrease in hepatic intrinsic clearance was present. For metoprolol, in contrast to propranolol, after IV administration, no changes in pharmacokinetic parameters as a result of inflammation were observed. After oral administration, the AUC was increased about 4 times in rats with inflammation; as metoprolol is only negligibly bound to serum proteins, the increase in AUC can be attributed to a decrease in hepatic intrinsic clearance.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukocyte Changes Associated with Acute Inflammation in Chickens

Leukocyte changes in chickens with turpentine-induced inflammation were investigated sequentially at 0, 6, 12, and 24 hours and at 2, 3, 4, 7, and 14 days. During acute inflammation, significant leukocytosis and heterophilia developed by 6 hours and persisted through 7 days. The peak mean heterophil and leukocyte counts occurred at 12 hours and 3 days, respectively. Left shifts were present at 12 and 24 hours as detected by 100-cell leukocyte differential counts. Heterophil mean nuclear scores documented nuclear hyposegmentation (left shift) during early inflammation and nuclear hypersegmentation (right shift) during convalescence. Mean monocyte and lymphocyte counts peaked at 2 and 3 days, respectively. Basophil and eosinophil counts were erratic. Toxic changes of heterophils were most apparent during intense left shifts and consisted of cell swelling, degranulation, cytoplasmic vacuolation, and cytoplasmic basophilia. Cytoplasmic basophilia was the last aspect of toxic change to resolve. Ultrastructurally, toxic heterophils had intracellular edema, dissolution of granules, retention of ribosomes, nuclear membrane blebs, and decreased heterochromatin density. All inflammation-associated alterations in cell counts and morphology returned to baseline values and appearance by 14 days after turpentine administration.

Transcriptional regulation of ceruloplasmin gene expression during inflammation.

Mixed sequence oligonucleotides were used to isolate a series of acute-phase human liver cDNA clones corresponding to the serum alpha 2-globulin ceruloplasmin. These clones were characterized, sequenced, and used to analyze changes in hepatic ceruloplasmin mRNA content during inflammation. In all species examined, hepatic ceruloplasmin mRNA content increased approximately 6-10-fold over control values within 24 h following the induction of inflammation. The mechanisms leading to this increase in hepatic ceruloplasmin mRNA content were studied following turpentine-induced inflammation in Syrian hamsters. Nuclear run-on assays demonstrated an increase in the relative rate of transcription of the ceruloplasmin gene within 3 h following induction, reaching maximum values by 18 h. Hepatic ceruloplasmin mRNA content increased 2-fold within 12 h following induction, reached maximum values by 24 h, and returned to control within 72 h. In contrast, serum ceruloplasmin concentration did not increase until 36 h, reached maximal levels by 120 h, and remained elevated for the course of the study. These data indicate that inflammation leads to a rapid increase in hepatic ceruloplasmin mRNA content. This increase is largely the result of increased ceruloplasmin gene transcription, but comparison of the relative rate of transcription and mRNA accumulation suggests that changes in ceruloplasmin mRNA turnover are also involved. In addition, translational and/or post-translational mechanisms must account for the observed changes in serum ceruloplasmin concentration seen during inflammation.

Influence of Copper Intake and Inflammation on Rat Serum Superoxide Dismutase Activity Levels

Serum superoxide dismutase (SOD) activity concentrations, though small compared to tissue levels, could contribute to extracellular superoxide radical detoxification and act as indicators of copper status. The present study identified the response of rat serum SOD activity contents to marginal and deficient copper intakes and to inflammation. Rats fed copper-deficient diet (less than 0.2 mg/kg Cu) for 5 wk displayed serum SOD activity contents that were only about 20% of those found in rats fed copper-supplemented diet (6.0 mg/kg Cu). Activities in rats fed a marginal diet (1.5 mg/kg Cu) were about 55% of those in the adequate rats. Turpentine-induced inflammation lowered serum SOD in rats within each dietary group. However, the change in the marginal group was not statistically significant. Based on chromatographic characterizations and inhibitor studies, rat serum SOD activity seemed to result primarily from a copper protein other than ceruloplasmin, Cu-Zn SOD or the recently discovered tissue extracellular SOD. In conclusion, low copper intake and inflammation may compromise extracellular defenses against superoxide. In addition, serum SOD activities could provide a non-ceruloplasmin-related means of assessing copper status, but nondietary variables can also affect these SOD values.

Glycosylation of chicken haptoglobin: Isolation and characterization of three molecular variants and studies of their distribution in hen plasma before and after turpentine-induced inflammation

Chicken haptoglobin (Hp), a hemoglobin-binding protein isolated from chicken plasma, is composed of three molecular variants that react differently with concanavalin A (ConA). These glycosylation variants of chicken Hp have been isolated by affinity chromatography using Sepharose-bound ConA. They differ in their molecular weight, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Analysis of the glycopeptides obtained after pronase digestion of these variants yielded two types of structures: one, reactive with ConA, corresponded to a biantennary N-linked carbohydrate unit and one, unreactive with ConA, corresponded to a triantennary unit. The strongly ConA-reactive Hp variant bears only two biantennary units and the nonreactive Hp variant bears only two triantennary units; the weakly reactive Hp variant bears equal amounts of both units. The distribution of Hp glycosylation variant does not show any significant difference when obtained from the plasma of laying hens before and after turpentine-induced inflammation.

Tissue distribution and kininogen gene expression after acute-phase inflammation

A kinin-directed monoclonal antibody to kininogens has been developed by the fusion of murine myeloma cells with mouse splenocytes immunized with bradykin-conjugated hemocyanin. The hybrid cells were screened by an enzyme-linked immunosorbent assay (ELISA) and a radioimmunoassay (RIA) for the secretion of antibodies to bradykinin. Ascitic fluids were produced and purified by a bradykinin-agarose affinity column. The monoclonal antibody (IgG 1) bound to bradykinin, Lys-bradykinin, Met-Lys-bradykinin, and kininogens in ELISA. Further, this target-directed monoclonal antibody recognized purified low and high molecular weight bovine, human, or rat kininogens and T-kininogen in Western blotting. After turpentine-induced acute inflammation, rat kininogen levels increased dramatically in liver and serum as well as in the perfused pituitary, heart, lung, kidney, thymus, and other tissues, as identified by the kinin-directed kininogen antibody in Western blot analyses. The results were confirmed by measuring kinin equivalents of kininogens with a kinin RIA. During an induced inflammatory response, rat kininogens were localized immunohistochemically with the kinin-directed monoclonal antibody in parenchymal cells of liver, in acinar cells and some granular convoluted tubules of submandibular gland, and in the collecting tubules of kidney. Northern and cytoplasmic dot blot analyses using a kinin oligonucleotide probe showed that kininogen mRNA levels in liver but not in other tissues increase after turpentine-induced inflammation. The results indicated that rat kininogens are distributed in various tissues in addition to liver and only liver kininogen is induced by acute inflammation. The target-directed kininogen monoclonal antibody is a useful reagent for studying the structure, localization, and function of kininogens or any protein molecule containing the kinin moiety.

Effect of inflammation on the metabolism of antipyrine, lidocaine and propranolol in isolated rat hepatocytes.

A decrease of the hepatic intrinsic clearance could contribute to the increase of the plasma concentrations of alpha 1-acid glycoprotein-bound drugs such as propranolol in animals and humans with inflammation. Therefore, the influence of inflammation upon the metabolism of propranolol and another high clearance drug, lidocaine, and of the low clearance drug antipyrine, was studied in isolated rat hepatocytes. For comparative purposes, the influence of the enzyme inhibitor SKF 525A (100 mg/kg i.p.) was also evaluated. Turpentine pretreatment of the rats significantly decreased the metabolism of the three drugs by the hepatocytes; the decrease was least pronounced for propranolol. The inhibitory effect of turpentine-induced inflammation was somewhat lower than that of SKF 525A. These results are in agreement with the results found for the same drugs in the 9,000-g supernatant fraction of the rat liver and point to a marked decrease of intrinsic clearance in some types of inflammation.

Rat corticotropin, insulin and thyroid hormone levels during the acute phase response to inflammation

1. 1. Circulating levels of corticotropin, thyroid hormones and insulin were measured in rats at various times after turpentine-induced inflammation. 2. 2. Corticotropin increased rapidly showing a biphasic response with a four-fold increase at about 6–8 hr after inflammation and a 10-fold increase at 10 hr after inflammation. 3. 3. The response of insulin to inflammation was slower than corticotropin and the magnitude of the increase was smaller. Insulin increased by three-fold at 20 hr after inflammation. 4. 4. Thyroid hormone levels were depressed by turpentine inflammation. Levels fell after 4hr and remained at low levels throughout. 5. 5. Administration of a cytokine preparation to rats also caused depressed thyroxine levels at short intervals after administration. However, levels increased at longer intervals after administration. 6. 6. This suggests that, like corticotropin and insulin, thyroid hormone levels could be under the control of immunotransmitters during the acute phase response.

Study of the molecular mechanism of decreased liver synthesis of albumin in inflammation.

Hypoalbuminemia in inflammatory disorders is not an infrequent finding. However, little is known about albumin synthesis in these patients. In the present study we have measured the albumin synthesis in four patients with inflammatory diseases using the [14C]carbonate technique. Because inflammation causes a decreased albumin synthesis and this decreased synthesis could not be related to a reduced amino acid supply, we have also examined the possible molecular mechanisms of reduced albumin synthesis during inflammation using in vivo and in vitro experiments in rats. In rats with turpentine-induced inflammation, serum albumin concentration and liver albumin mRNa level were markedly decreased. These changes could not be reproduced by administration of fibrinogen-, or fibrin-degradation products, or several hormones, such as corticosteroids, growth hormone, and adrenaline. However, monocytic products, especially interleukin 1, postulated to be important mediators of the inflammatory response, reduced albumin synthesis and liver albumin messenger RNA content but not total protein synthesis in rats in vivo and in primary cultures of rat hepatocytes. These findings suggest that monocytic products play an important role in reduced albumin synthesis during inflammation.

Effects of ascorbic acid and inflammation on ceruloplasmin activity levels in guinea pigs

The possibility that ascorbic acid exerts a postabsorptive effect on copper metabolism in guinea pigs was examined. A pair of ascorbic acid injections (20 mg/kg, ip, 18 h apart) caused a small but statistically significant increase in oxidase activity levels of ceruloplasmin, a serum copper protein. In contrast, dietary ascorbic acid deprivation for 14 days did not prevent increases in ceruloplasmin activity levels caused by turpentine-induced inflammation. The ascorbic acid deprivation did reduce weight gain and adrenal ascorbic acid contents but serum ascorbic acid concentrations were not significantly lowered. In summary, ascorbic acid can produce postabsorptive changes in copper metabolism but adequate intake of the vitamin is not necessary for inflammation-induced elevations of ceruloplasmin activity levels.

Inflammation-associated events in liver nuclei during acute-phase reaction

Isolated liver nucleoli from rats undergoing turpentine-induced inflammation (acute-phase reaction) synthesize rRNA at a rate significantly higher than normal. This increase is associated with, and possibly preceded by, an enhanced methylation of RNA, which further increases when rRNA synthesis has reached a plateau level. Five hours after turpentine treatment, before clear activation of RNA synthesis and methylations, the nucleocytoplasmic transport of rRNA (largely 40S and 60S subunits) and the related ATPase activity of isolated nuclei are significantly increased. Apparently, posttranscriptional control is affected before transcription of rRNA during the onset of the acute-phase reaction: both kinds of events eventually contribute to the expansion of the ribosome population which occurs in the liver cells from rats undergoing an inflammatory process. All these processes are activated before the liver starts the synthesis of acute-phase proteins.

Mouse α 1-protease inhibitor is not an acute phase reactant

Mouse plasma contains two major protease inhibitors, α 1-protease inhibitor (α 1-PI) and contrapsin, which have high affinity for bovine trypsin. Systemic injury, such as turpentine-induced inflammation, did not change the plasma concentration of α 1-PI, but increased that of contrapsin by 50%. The concentration of hepatic α 1-PI mRNA was determined by Northern blot hybridization and was not significantly affected by the acute phase reaction. J. M. Frazer, S. A. Nathoo, J. Katz, T. L. Genetta, and T. H. Finley ((1985) Arch. Biochem. Biophys. 239, 112–119) have reported a threefold increase of mRNA for the elastase specific α 1-PI but this increase was not demonstrated by the present study. The mRNAs for known mouse acute phase plasma proteins were, however, stimulated severalfold by the same treatment. These results indicate that in the mouse, as opposed to human, α 1-PI is not an acute phase reactant.

Preventive Effects of Acute Inflammation on Liver Cell Necrosis and Inhibition of Heparan Sulphate Synthesis in Hepatocytes

The hepatocytoprotective effect of acute, turpentine-induced inflammation on experimental liver injury, caused by thioacetamide and D-galactosamine, respectively, was studied in relation to the synthesis of glycosaminoglycans and of heparan sulphate in hepatocytes isolated from livers of treated rats. As judged from biochemical parameters of liver cell lesion in serum (various cytosolic and mitochondrial enzymes, bile acids) the extent of liver damage caused by either noxious agent was greatly attenuated under acute inflammatory conditions. There was an inverse statistical correlation (r = -0.63 to r = -0.84) between the functional concentration of a proteinase inhibitor protein determined with the chromogenic substrate assay for human alpha 2-macroglobulin and the catalytic concentrations of various cell leakage enzymes in serum from liver-injured rats with turpentine-generated inflammation. The strong inhibition of heparan sulphate synthesis in hepatocytes from injured livers (synthesis rate between 0.2 and 0.4 of that in control cell incubations) is abolished in hepatocytes from livers exposed to the noxious agents but with acute inflammation. The latter condition alone caused a 1.8 fold increase in heparan sulphate synthesis of hepatocytes. Heparan sulphate was the only type of glycosaminoglycan synthesized under all conditions. The results are discussed in view of the pathogenetic potential of heparan sulphate for liver cell necrosis.

Transcortin and α 2u-globulin messenger rna activities during turpentine-induced inflammation in the rat

Previously we have shown that the serum concentration of transcortin and α 2u-globulin markedly decreases during turpentine-induced inflammation. In the present study transcortin and α 2u-globulin mRNA from healthy rats and from animals with inflammation was translated in a rabbit reticulocyte lysate system. Female rats had higher levels of translatable transcortin mRNA than male animals and the level of mRNA for transcortin and α 2u-globulin decreased rapidly during inflammation. These results indicate that the sex difference in the serum level of transcortin and the changes in serum transcortin and α 2u-globulin during inflammation are mainly determined by differences in the mRNAs in the liver.

Changes of the level of proteinase inhibitors in rat plasma during turpentine-induced inflammation.

The levels of rat plasma alpha-macroglobulins, alpha-cysteine proteinase inhibitor, haptoglobin and antipapain activity were studied during the acute-phase reaction after an injection of alpha-pinen. An increase in concentration of all the compounds examined was observed.

Inflammation-induced increases in dolichol synthesis and hydroxymethylglutaryl-coenzyme a reductase activity in mouse liver are prevented by a high-cholesterol diet but not by fasting

The inflammatory response in mammals is characterized by the synthesis in the liver of several N-linked serum glycoproteins called acute-phase reactants. In C57BL/6J mice, turpentine-induced inflammation was accompanied by increases in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, dolichol synthesis, and dolichyl phosphoryl mannose synthesis. Cholesterol feeding, but not fasting, prevented these inflammation-induced increases in reductase activity and dolichol synthesis. However, the rate of incorporation of [ 3H]mannose into total serum glycoproteins was not affected by the high-cholesterol diet, and this rate increased during acute inflammation in control and cholesterol-fed mice.

Inflammation triggers hypoferremia and de novo synthesis of serum transferrin and ceruloplasmin in mice.

Oil of turpentine was used to induce an artificial inflammation so that we could study its effect on iron metabolism and on synthesis of serum transferrin and ceruloplasmin in mice. It was found that turpentine-induced inflammation triggered the establishment of a hypoferremic state characterized by low levels of serum iron, followed by recovery and a gradual return to normal plasma iron levels. This turpentine-induced hypoferremia and its subsequent recovery paralleled the hypoferremia obtained during meningococcal infection. Moreover, serum transferrin and ceruloplasmin activity levels increased drastically during the recovery from hypoferremia. [14C]leucine incorporation studies revealed a de novo synthesis of both transferrin and ceruloplasmin. Turpentine-induced hypoferremia was also found to provide a protective effect against meningococcal infection which could be partially reversed by exogenous iron. The results of this study suggest that transferrin and ceruloplasmin may be synthesized partly in response to the altered iron metabolism observed during hypoferremia.

Acute local inflammation alters synthesis, distribution, and catabolism of third component of complement (C3) in rabbits.

In order to evaluate the basis for changes in plasma concentrations of the third component of complement (C3) during inflammation, we injected purified radiolabeled C3 into normal New Zealand White rabbits and into rabbits with turpentine-induced pleurisy. In the normal animals, C3 was distributed between the intravascular compartment (75%) and the extravascular space (25%), with an exchange rate of 1.8 +/- 0.1% of the plasma pool per hour. The fractional catabolic rate (FCR) was 2.7 +/- 0.3% of the C3 plasma pool per hour, the synthesis rate was 1.0 +/- 0.2 mg C3/kg per h, and the plasma concentration was 1.23 +/- 0.3 mg C3/ml. Rabbits with turpentine-induced inflammation showed a shift of the volume of C3 distribution in favor of the extravascular compartment. In addition, the rate by which 125I-C3 was cleared from the circulation increased by 29% and was related to the appearance of 20% of the C3-bound circulating radioactivity in the affected pleural cavity at the zenith of inflammation. The FCR, calculated by measuring urinary excretion of radiolabel, increased by only 9% and was probably related to the C3 degradation that was observed in the pleural fluid during the early stages of inflammation. The plasma C3 concentration reached a peak at 230% of the baseline concentration, owing to an increase in the rate of synthesis by as much as 480%. The latter increase could be blocked by cycloheximide, an inhibitor of protein synthesis. We conclude that the increase of plasma C3 in the acute phase is due to stimulated synthesis, which is partially offset by a rise in FCR and by a shift of protein to the site of inflammation.

Isolated hepatocytes fixed on collagen, an accurate model for the the secretion of proteins in a minimal medium. Response to inflammation

The albumin, orosomucoïd and alpha 2-macroglobulin secretion by isolated hepatocytes of normal and suffering from Turpentine-induced inflammation rats, is investigated for 4 hr. The model, stable over the whole duration of incubation, is a true reflect of hepatic secretion in vivo and can be used to measure it.