Hepatic metallothionein induction in inflammation.

Numerous chemically distinct phlogistic substances have been shown to induce hepatic metallothionein-Zn (MT) accumulation when administered to rats. These findings suggest that induction of this cysteine-rich metalloprotein occurs through the action of some common mediator(s). Possible mediators include substances such as leukocytic endogenous mediator (LEM) and/or hormones known to influence hepatic protein synthesis. Studies were performed to examine further the mechanism(s) and potential mediators involved in endotoxin-induced MT accumulation. Additionally, the studies were performed to determine the possible involvement of genetic factors, which reportedly influence LEM production, in the induced MT response. Endotoxin (ET) was administered ip to rats and to EP-resistant, C3H/HeJ, and susceptible, C3Heb/FeJ, stains of mice. ET induced hypozincemia, hyperglucagonemia, and increased MT concentrations in rats. ET induced hypozincemia and MT accumulation to the same extent in both strains of mice. The induction of tolerance in rats to Zn depressing activity of ET also prevented hyperglucagonemia and additional accumulation of MT. Results suggest that glucagon, but not LEM, may be a common mediator in MT response during inflammatory stress.

Effect of adrenalectomy on cadmium- and turpentine-induced hepatic synthesis of metallothionein and alpha 2-macrofetoprotein in the rat.

Recent reports have strongly implicated glucocorticoids in the induction of hepatic metallothionein synthesis and hypozincemia which occurs in certain pathophysiologic conditions. Studies were performed in rats to determine the effect of adrenalectomy and glucocorticoid treatment on the hepatic accumulation of metallothionein subsequent to the administration of cadmium and turpentine, two diverse substances known to induce hypozincemia and hepatic synthesis of metallothionein as well as alpha 2-macrofetoprotein in intact rats. By 24 h, both substances induced significant hypozincemia, hepatic metallothionein accumulation, and a severe tissue inflammatory response in adrenalectomized rats. Adrenalectomy only prevented the increase in plasma alpha 2-macrofetoprotein concentration. Results indicate that hepatic synthesis of alpha 2-macrofetoprotein, but not metallothionein, is mediated by adrenal hormones. Thus, glucocorticoids do not play a "vital" role in hepatic metallothionein accumulation or hypozincemia induced by inflammatory stress, as previously postulated.

Differential alterations in host peripheral polymorphonuclear leukocyte chemiluminescence during the course of bacterial and viral infections.

Previous studies have shown that stimulation of the oxidative metabolism in polymorphonuclear leukocytes (PMN) by in vitro phagocytosis of various microorganisms results in photon emission, termed chemiluminescence (CL). Studies were conducted to determine whether bacterial and viral infections induce enhanced basal endogenous host peripheral PMN CL in the absence of in vitro phagocytic stimulation. Nonimmune rats and guinea pigs as well as immune rats were inoculated with various doses (10(5) to 10(7)) of live vaccine strain Francisella tularensis (per 100 g of body weight). In addition, nonimmune guinea pigs were inoculated with 40,000 plaque-forming units of Pichinde virus. Luminol-assisted endogenous PMN CL was measured at various time intervals after inoculation of microorganisms. Enhanced endogenous PMN CL was detected as early as the appearance of fever (12 h) in nonimmune animals infected with F. tularensis. Addition of sodium azide, N-ethylmaleimide, superoxide dismutase, or catalase to the CL reaction mixture containing PMN from infected animals significantly decreased the CL response. Immune rats challenged with F. tularensis exhibited resistance to infection and a decreased PMN CL compared with nonimmune rats 24 and 48 h after inoculation. However, the CL response from immune rats was significantly elevated, compared with control values. In contrast to the results obtained with the model bacterial infection, PMN isolated from guinea pigs inoculated with Pichinde virus failed to exhibit enhanced CL, compared with controls, despite significant viremia and fever. Results suggest that enhanced endogenous CL during bacterial infection occurs through mechanisms involving increased PMN oxidative metabolism and the subsequent generation of microbicidal forms of oxygen. Further, measurement of endogenous PMN CL may have diagnostic and prognostic value in infectious diseases.

Protection of dogs from lethal consequences of endotoxemia with plasma or leukocyte transfusions.

The contribution of cellular and humoral factors of normal blood to resistance to endotoxin was evaluated by selectively transfusing them into beagle dogs prior to IV challenge with a lethal (2.75 mg/kg) dose of Salmonella typhi endotoxin. Supplementation of normal host defenses with 250 ml of plasma containing a heatlabile (56 degrees C for one hour) factor protected the dogs from lethal effects of the toxin. A similar volume of heparinized saline or a lesser volume of plasma (100 ml) was ineffective. The presence of 1 X 10(9) platelets and 7 X 10(10) leukocytes from leukapheresed foxhounds in some transfusion preparations did not affect survival. Protection by treatment with plasma was accompanied by severe tissue injury and loss of circulating platelets and leukocytes. Granulocyte concentrates also afforded protection and decreased tissue injury, as indicated by SGPT and taurine levels. Survivor and nonsurvivor animals experienced an early hyperglycemia as well as elevation of serum glutamic pyruvic transaminase and taurine levels. Thrombocytopenia was great in all experimental groups but was less marked in dogs transfused with cells. Leukopenia was comparable in all groups until six hours after challenge, at which time numbers of circulating leukocytes began a significant return toward normal levels in the cell-transfused group. Impairment of macrophage function was indicated by the depression of the release of colony-stimulating factor in survivor and nonsurvivor animals. Thus, normal plasma alone can protect dogs from endotoxin, but not without a significant amount of injury to the host.

Effects of intravenous injection of leukocytic endogenous mediator on cardiohepatic functions in rhesus macaques.

A crude preparation of leukocytic endogenous mediator administered IV over a 10-minute period into a rhesus macaque at a dosage of 10 ml/kg of body weight resulted in hypotension, tachycardia, vasodilation, hypoalbuminemia, and hypoproteinemia. Decreases in cardiac and hepatic functions and biphasic changes in peripheral polymorphonuclear leukocytes also were seen. All measured changes, except hepatic functions and plasma albumin values, returned to base-line values within 24 hours. Data indicate that presently utilized crude leucocytic endogenous mediator preparations contain a heretofore undescribed, and as yet unidentified, component. Induced early cardiovascular changes may be related in part to certain compounds likely to be in the crude preparation.

Possible association of granulocyte mobilization to the peritoneal cavity with ZnCl2-induced protection against endotoxin.

We have attempted to determine which components of the inflammatory response are responsible for ZnCl2-induced retention of endotoxin in the peritoneal cavity and enhancement of survival following challange with the toxin. ZnCl2 injected intraperitoneally into mice caused accumulation of granulocytes in the peritoneal cavity, but these cells were apparently not responsible for the trapping process. This contention in supported by our observation that reduction of hepatosplenic uptake of 51CR-labeled endotonxin was similar in unirradiated mice and in mice made by irradiation (1000 rad 60 Co) 1 rad = 10 (-2) J/kg). Hepatosplenic uptake was also depressed when untreated mice were injected with endotoxin suspended in cell-free plasma. Furthermore, zinc did not protect irradiated mice challanged with endotoxin, although it enhanced survival in urirradiated animals. Lack of protection in irradiated mice may be due to a deficiency in the cellular response in the peritoneal cavity.

Involvement of hepatic metallothioneins in hypozincemia associated with bacterial infection.

Hypozincemia was induced in rats by Salmonella typhimurium and live vaccine strain Francisella tularensis (LVS) infections. Hepatic synthesis of zinc-binding proteins (ZBP) was studied in order to elucidate the mechanisms involved in the redistribution of zinc from plasma to liver occurring during infectious illness. ZBP, labeled in vivo with 65Zn, were isolated and identified as metallothioneins based, in part, on their heat stability, dimorphism, and amino acid composition. Cysteine was the major amino acid found in both forms of metallothionein and constituted 28-31% of total residues. The apparent half-life of these proteins as measured by disappearance of 65Zn was determined to be 19 h in a relatively mild infection (LVS) and 38 h in a more severe S. typhimurium infection. Results provide evidence that metallothioneins not only have the previously postulated regulatory role in normal zinc homeostasis but are intimately involved in the zinc redistribution occurring during the acute stage of infectious illness.

In vitro and in vivo actions of zinc ion affecting cellular substances which influence host metabolic responses to inflammation.

Glycogen-stimulated rabbit peritoneal exudate cells (polymorphonuclear leukocytes, PMN) produce prostaglandins (PG) and substances which induce alterations (mediators) in experimental animals characteristic of host metabolic responses to infection and other acute inflammatory stresses. The effect of Zn2+ on mediator production and PG synthesis was examined because: Zn homeostasis is perturbed during infection, Zn is known to regulate some cellular functions, and there appears to be an interrelationship between PG synthesis and mediator production. Using exudate cells, 2 mM Zn2+ caused complete inhibition of in vitro PG synthesis as assessed by conversion of [1-14C] arachidonic acid into PG. This concentration of Zn2+ also inhibited production of substances mediating plasma Zn depression, hepatic amino acid "uptake", fever, and neutrophil release from bone marrow. Conversely, Zn2+ did not inhibit in vivo metabolic responses to these mediators. Zn-pretreatment of rabbits or simultaneous injection of Zn2+ and crude PMN-derived pyrogenic activity resulted in prolongation of fever. It is suggested that this action of Zn2+ may be attributed to either stabilization of cyclic AMP through inhibition of phosphodiesterase or a Zn-mediator interaction which stabilizes crude endogenous pyrogen. The potential physiological significance of these results includes: possible potentiation of the host's defense mechanisms by Zn2+ and its utilization for prolongation of fever to determine its effect on potentially temperature-dependent host defense mechanisms.

Effect of leukocytic endogenous mediators on endocrine pancreas secretory responses.

Crude mediators from stimulated rabbit peritoneal leukocytes (LEM) engender numerous physiologic alterations in rats, which are similar to those observed during infection. One hour after the intraperitoneal injection of crude LEM, plasma insulin and glucagon concentrations are elevated; at 2 h the hormonal alterations are manifested by a 30% increase in hepatic cyclic adenosine 3',5'-monophosphate (cAMP), glycogen depression, and uptake of 14C-labeled nonmetabolizable amino acid analogues (AA). Plasma hormone concentrations reach maximum levels by 5 h and decline by 24 h. The hepatic concentrations of AA parallel the insulin and glucagon responses and correlate with the inverse of insulin/glucagon molar ratio. In spite of mobilization of hepatic glycogen evident at 5 h, plasma glucose concentrations were transiently depressed. Plasma insulin, glucagon, and hepatic AA concentrations were dose dependent. Plasma insulin and glucagon responses to crude LEM may explain increases in hepatic cAMP, uptake of AA, and glycogenolysis as well as hypoglycemia. These data partially characterize the role of crude LEM, provide an explanation for the stimuli-inducing hyperglucagonemia and hyperinsulinemia during infection. They implicate the endocrine pancreas as a factor regulating the host's metabolic response to infection.

Role of zinc in the abatement of hepatocellular damage and mortality incidence in endotoxemic rats.

Intraperitoneal administration of zinc (ZnIP) as zinc chloride prior to or simultaneously with a lethal quantity of intraperitoneally administered Salmonella typhimurium endotoxin significantly protected rats against toxin-induced mortality and hepatocellular damage. Pretreatment with amounts of zinc chloride ranging from 0.4 to 2.0 mg/100 g of body weight resulted in 80 to 100% survival compared with 10% survival in untreated control rats at 24 h after endotoxin treatment. Zinc chloride treatment in excess of 2.0 mg/100 g of body weight appeared to be toxic and provided diminished protection. In contrast with the protection obtained with ZnIP, intravenously administered zinc did not provide protection. The effectiveness of ZnIP to enhance survival if it was given after endotoxin was greatly diminished as a function of time after endotoxin. The extent of hepatocellular damage was assessed at various times after endotoxin administration in ZnIP-treated and untreated rats by measurement of plasma ornithine carbamoyltransferase activity and histological examination of liver sections. Endotoxin absorption from the peritoneal cavity and hepatic uptake were studied by using 51Cr-labeled endotoxin. ZnIP pretreatment significantly reduced 51Cr-labeled endotoxin content of blood and liver when compared to untreated controls, and effectively prevented endotoxin-induced elevations in plasma ornithine carbamoyltransferase activity and hepatic tissue necrosis. These data indicate that protection afforded by ZnIP treatment results as a consequence of the ability of zinc to diminish absorption of the toxin from the peritoneal cavity and subsequent hepatic uptake.

Possible relation of prostaglandins to PMN-derived mediators of host metabolic responses to inflammation.

Stimulated rabbit peritoneal polymorphonuclear leukocyte (PMN) preparations simultaneously produce prostaglandin-like material and mediators that induce metabolic alterations in experimental animals characteristic of the host's responses to inflammation. The alterations observed in rats include responses by: proteins, carbohydrates, hormones, trace metals, and total blood neutrophils. This study demonstrates a possible relationship between prostaglandins and PMN-derived substances that mediate plasma zinc depression, hepatic amino acid uptake, and increased numbers of blood neutrophils. Production of these mediators by stimulated-PMN preparations was prevented by 23 muM indomethacin or 93 muM aspirin. Conversely, morphine (2 mM or less) had no detrimental effect on production of these mediators, although, it consistently stimulated production of a substance stimulating total blood neutrophia. In addition, 2 muM prostaglandin E and F stimulated production of substances mediating hepatic amino acid uptake plasma zinc depression, respectively. At this concentration, neither prostaglandin significantly altered production of substances mediating increased numbers of total blood neutrophils. A partial-nitrogen atmosphere, dibutyryl cyclic analogs of AMP and GMP, or homogenization of the PMN had no effect on mediator production. The inhibitory effect of indomethacin and aspirin also was observed with PMN-homogenastes. These experimental observations suggest that prostaglandin synthesis has a function in production of mediators by stimulated-PMN preparations.

Differentiation between endogenous pyrogen and leukocytic endogenous mediator.

The crude material released from glycogen-stimulated rabbit peritoneal polymorphonuclear leukocytes when administered to experimental animals elicits a number of metabolic and physiologic alterations characteristic of those observed in the host inflammatory response. Classically, the mediator of febrile response observed in rabbits and other species has been termed endogenous pyrogen (EP), whereas leukocytic endogenous mediator (LEM) has been used as a general term to denote the substance(s) mediating multiple inflammatory responses observed in rats. The latter substance, however, has not been previously demonstrated to differ from EP. This report presents evidence indicating that EP and LEM are different molecular species. Evidence supporting the differentiation between these entities includes: physical separation of EP from one or more mediators that induce metabolic alterations attributed to LEM; production of LEM activities by stimulated polymorphonuclear leukocytes in the absence of detectable pyrogenic activity; and differences in the release of EP and LEM from stimulated rabbit granulocytes in the presence of potassium ion.

Improved continuous-flow method for detemination of total serum hexosamines.

We describe an automated determination of serum hexosamine by the Elson-Morgan reaction, together with reagent modifications that minimiz interference from amino acids and sugars present in acid hydrolysates of sera. We used a novel 15-min autoclave procedure for the acid hydrolysis of sera before analysis to facilitate the detemination as compared to the 4-h hydrolysis used in the conventional manual method. Results correlated well (r = 0.906) with those obtained by the corresponding manual method.

Chronic depression of serum concentrations of sialic acid in alloxan-induced diabetic rats.

This study was performed to determine whether alloxan treatment of rats alters concentrations of the terminal carbohydrate residues, L-fucose and sialic acid, of serum glycoproteins. Results indicate that in the uncompensated diabetic rat, chronic depression of serum sialic acid concentration occurred with no apparent alteration in the concentration of L-fucose. The depression in sialic acid concentration may be attributed, in part, to decreased activities of hepatic enzymes involved in sialic acid synthesis similar to those observed by others after treatment of rats with the diabetogenic agent streptozotocin. The lack of any significant alteration in the concentration of L-fucose fails to confirm, in the experimental diabetic rat, the increased protein-bound fucose values reported in human diabetics. Administration of insulin was not effective in modifying the sialic acid response after alloxan treatment.