metallothionein and Acute-Phase-Reaction

A multifunctional protein metallothionein (MT) is induced by various chemicals and cytokines. We have found novel functions of MT as follows: 1) Cytokine expression such as IL-1alpha, IL-6, and TNFalpha responding to lipopolysaccharide is reduced in MT-deficient macrophages compared with in wild-type cells. 2) Nitric oxide production responding to TNFalpha and LPS is reduced in MT-deficient macrophages compared with in wild-type cells. 3) M-CSF expression responding to zinc is reduced in MT-deficient fibroblasts compared with in wild-type cells, and increased in MT-overexpressed fibroblasts compared with in control cells. 4) LIF, a STAT3 activating cytokine, protects the heart from ischemia/reperfusion injury. Transgenic mice overexpressing STAT3 have tolerance to ischemia/reperfusion-induced damage, whereas MT-null mutation cancels the myocardial protection. In this review, we discuss the relation of MT and stress responses from the point of view of cytokine-induced expression of MT and modulation of cytokine expression by MT.

Topics: Acute-Phase Reaction; Animals; Cytokines; Fibroblasts; Interleukin-6; Macrophage Colony-Stimulating Factor; Macrophages; Metallothionein; Mice; Myocardial Reperfusion Injury; NF-kappa B; STAT3 Transcription Factor; Tumor Necrosis Factor-alpha

In experimental studies on the common human coxsackievirus B type 3 (CB3) infection, administered cadmium (Cd) is known to accumulate in the liver and kidneys. CB3 adapted to Balb/c mice was used to study whether infection affects the Cd-binding protein, metallothionein (MT) and if this alters the normal physiological trace element balance in the liver, kidney, spleen and brain. On day 3 of infection, degradation of liver proteins (44%, P<0.01) occurred, whereas in the spleen, protein increased (63%, P<0.05). The infection increased MT five-fold (P<0.01) in liver and kidneys, and in spleen by 34% (P<0.05). A redistribution of Cd and copper (Cu) from the liver to the kidney was associated with this increase in MT, resulting in an increased (P<0.01) kidney/liver ratio for both elements. The infection increased the zinc (Zn) concentration more in the kidney than in the liver, but the kidney/liver ratio was not significantly affected. Results show that MT is increased in several organs during the early phase of infection and is associated with redistribution of both essential and non-essential trace elements. This may be a normal response in common infections that could adversely influence the pathogenesis when the host is concomitantly exposed to potentially toxic trace elements, even at levels in the physiological range.

Topics: Acute-Phase Reaction; Animals; Brain; Cadmium; Copper; Disease Models, Animal; Enterovirus B, Human; Enterovirus Infections; Female; Kidney; Liver; Metallothionein; Mice; Mice, Inbred BALB C; Organ Size; Proteins; Spleen; Trace Elements; Zinc

Lipopolysaccharide (LPS) is embryolethal in CD-1 mice. LPS induces metallothionein (MT) via cytokines, including TNF-alpha, IL-1, and IL-6, which initiate and maintain the acute phase response. Maternal hepatic MT induction in pregnant rats, by diverse toxicants, can result in maternal hypozincemia and subsequent embryonal zinc (Zn) deficiency. We examined the hypothesis that LPS causes embryo toxicity in CD-1 mice via MT induction and subsequent embryo Zn deficiency by (1) determining whether LPS induces maternal hepatic MT and causes Zn redistribution, (2) assessing the effects of maternal Zn supplementation on LPS developmental toxicity, and (3) assessing the role of MT with MT I-II null mice (MTKO). Timed pregnant CD-1 mice were dosed i.p. with LPS (S. typhimurium) (0.05 mg/kg) on gestation day (gd) 9. Zn supplementation was administered on gd 8 (10 mg/kg, pretreatment) or on gd 9 as a cotreatment (5 or 10 mg/kg). MTKO and wild type (WT) mice were dosed with LPS (0.05 or 0.1 mg/kg) on gd 9, and maternal liver MT and Zn and plasma Zn were measured. In CD-1 mice, maternal hepatic MT was elevated 24 h after LPS treatment, and cotreatment with Zn caused further elevation of MT. Maternal hepatic Zn concentrations paralleled hepatic MT concentrations. Maternal plasma Zn on gd 10 showed no consistent effect of LPS treatment or Zn cotreatment on gd 9. Zn pretreatment (10 mg/kg) on gd 8 did not ameliorate LPS embryolethality, while Zn cotreatment (5 or 10 mg/kg) on gd 9 exacerbated the toxicity of LPS. LPS produced a similar incidence of embryolethality in MTKO and WT strains on gd10. Plasma Zn concentrations were similar in both strains, while hepatic Zn concentrations were significantly higher in WT than in the MTKO strain. In conclusion, while LPS can induce maternal hepatic MT and Zn redistribution in CD-1 mice, this does not appear to be a key mechanism leading to LPS embryotoxicity.

Topics: Acute-Phase Reaction; Animals; Drug Synergism; Female; Fetal Death; Gestational Age; Injections, Intraperitoneal; Lipopolysaccharides; Liver; Metallothionein; Mice; Mice, Inbred Strains; Mice, Knockout; Pregnancy; Salmonella typhimurium; Species Specificity; Zinc

We used a microarray technique to investigate changes of gene expression in liver induced by two peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, a strong PPARalpha agonist, Wy-14,643, and a marketed fibrate drug, fenofibrate. The purposes of this work are: 1) to examine whether or not gene expression is altered in different ways by these two PPARalpha agonists and 2) to find genes whose expression has not been previously reported to be affected by PPARalpha agonists. Mice were treated orally with 100 mg/kg fenofibrate, or 30 mg/kg or 100 mg/kg Wy-14,643, and the liver was collected on Day 2 or 3. mRNA was extraction from liver, and subjected to microarray analysis. Previously reported induction or reduction of gene expression, e.g. genes involved in beta-oxidation and lipid metabolism, was confirmed in our study. Scatter plot analysis indicated that the changes of gene expression pattern induced by fenofibrate and Wy-14,643 were almost identical. However, expression levels of metallothionein 1 and 2 mRNAs were different: no change of hepatic metallothionein 1 and 2 mRNA expression was induced by 100 mg/kg fenofibrate on Day 2 or 3, while 30 mg/kg Wy-14,643 administration increased expression of both genes by 1.8-fold on Day 3. In addition to previously reported gene expression changes by PPARalpha agonists, we found expression changes of other genes, including cis-retinol/3alpha-hydroxysterol short chain dehydrogenase, vanin-1, RecA-like protein, and serum amyloid A (SAA) 2. Among them, the change of SAA2 mRNA level was noteworthy; it showed a decrease to as little as one-seventh. Seven-day fenofibrate pre-treatment of mice completely inhibited the acute-phase elevation of plasma SAA concentration triggered by acetaminophen challenge. This finding suggests that fenofibrate treatment may reduce plasma SAA concentration in patients with secondary amyloidosis.

Topics: Acute-Phase Reaction; Animals; Down-Regulation; Fenofibrate; Gene Expression Profiling; Liver; Metallothionein; Mice; Mice, Inbred C57BL; Oligonucleotide Array Sequence Analysis; Pyrimidines; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Serum Amyloid A Protein; Transcription Factors

The serum amyloid A (SAA) family of proteins consists of inducible acute-phase members and a constitutive member that are minor apolipoproteins of normal high density lipoprotein (HDL). During inflammation, HDL cholesterol and apolipoprotein A-I (apoA-I) protein are decreased, and these changes are thought to be partly related to the increase in acute-phase SAA proteins that associate with the HDL particle to become the major apolipoprotein species. To determine the specific role of SAA in the alteration of HDL in the absence of a generalized acute-phase response, acute-phase Saa1.1 transgene expression was directed via an inducible mouse metallothionein promoter. Elevated levels of SAA1.1 (28+/-9 mg/dL) comparable to a moderate acute-phase response were achieved over a 5-day period. SAA association with the HDL particles at this concentration did not significantly alter the apoA-I or HDL cholesterol levels or change the lipoprotein profiles in the transgenic mice compared with wild-type mice. In addition, we used adenoviral vectors to increase the SAA expression to levels seen in a major acute-phase response. Injection of adenovirus expressing the mouse SAA1.1 protein resulted in high-level expression (72+/-8 mg/dL) but did not alter apoA-I levels. However, the SAA associated with the HDL particle gave rise to significantly larger HDL particles ( approximately 10%). Adenoviral expression of the constitutive SAA4 protein resulted in an increase in HDL size ( approximately 10%) and an increase in very low density lipoprotein levels (20-fold) and triglyceride levels (1.7-fold). These studies suggest that increases in acute-phase SAA proteins alone are insufficient to alter HDL cholesterol or apoA-I levels during inflammation. A role for constitutive SAA4 in HDL-very low density lipoprotein interactions should be considered.

Topics: Acute-Phase Reaction; Adenoviridae; Animals; Apolipoprotein A-I; Cholesterol, HDL; Gene Expression; Genetic Vectors; Lipoproteins; Lipoproteins, HDL; Metallothionein; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred ICR; Mice, Transgenic; Promoter Regions, Genetic; RNA, Messenger; Serum Amyloid A Protein; Zinc Sulfate

Cysteine-rich intestinal protein (CRIP), a double zinc-finger LIM protein, is expressed in great abundance in the intestine. We have found comparable levels of CRIP mRNA in peritoneal macrophages, peripheral blood mononuclear cells (PBMC), and lesser amounts in thymus and spleen. Because CRIP expression was high in immune cells, rats were challenged with lipopolysaccharide (LPS) to determine whether expression was altered during the acute-phase immune response. Immunocytochemistry showed that, in adherent mononuclear cells, CRIP protein was localized in the cytoplasm. CRIP mRNA levels increased over time after LPS injection in peritoneal macrophages, PBMC, spleen, and intestine. No changes in CRIP mRNA level were seen in either liver or thymus. In PBMC, the level of CRIP mRNA decreased before increasing later in the acute-phase immune response. CRIP protein was found in the plasma. Shortly after LPS administration plasma CRIP decreased, suggesting that CRIP was either passively diffused out of capillaries or was actively shunted into tissues to execute its function. Increased CRIP expression seen in response to LPS suggests that CRIP may play a role in immune cell activation or differentiation or in processes associated with cellular repair.

Topics: Acute-Phase Reaction; Animals; Blotting, Western; Carrier Proteins; Intestinal Mucosa; Intestines; Leukocytes, Mononuclear; LIM Domain Proteins; Lipopolysaccharides; Liver; Macrophages, Peritoneal; Male; Metallothionein; Rats; Rats, Sprague-Dawley; RNA, Messenger; Zinc; Zinc Fingers

The hepatic response to injury is orchestrated by the expression of different gene groups (i.e., heat shock and acute phase). In the present study, the expression of heat shock and acute phase genes was analyzed in the context of a localized injury, regional hepatic ischemia-reperfusion. Left and median liver lobes were subjected to 1 h of ischemia, whereas blood flow was maintained to the remainder of the organ. After the period of ischemia, the organ was reperfused, and samples of the ischemic and nonischemic liver were obtained at different time points during reperfusion. Expression of the heat shock gene, HSP 72, was detected only in the ischemic liver, whereas expression of the acute phase gene, beta-fibrinogen, and the interleukin-6-inducible gene, metallothionein, was maximally induced in the nonischemic liver and attenuated in the ischemic liver. To determine how the heat shock and acute phase responses were reprioritized during stress, expression of beta-fibronogen and HSP 72 was induced simultaneously in the same animal by administration of endotoxin and total body hyperthermia, respectively. Administration of endotoxin did not impede the expression of HSP 72; however, heat shock attenuated, but did not eliminate, the endotoxin-induced expression of beta-fibronogen. These observations suggest that the heat shock and acute phase responses are not mutually exclusive.

Topics: Acute-Phase Proteins; Acute-Phase Reaction; Animals; Fibrinogen; Gene Expression; Heat-Shock Proteins; Heat-Shock Response; HSP72 Heat-Shock Proteins; Ischemia; Liver; Liver Circulation; Male; Metallothionein; Phosphoenolpyruvate Carboxykinase (GTP); Rats; Rats, Sprague-Dawley; Reperfusion; RNA, Messenger

The development of a sensitive enzyme-linked immunosorbent assay (ELISA) for human metallothionein-1 is reported. Metallothionein was purified from postmortem human liver and used to raise high-titer antibodies in rabbits. The assay was specific for human metallothionein-1 (MT-1), and there was no significant cross-reaction with human metallothionein-2. The detection limit (sensitivity) of the assay was 5 ng/ml, and the added MT-1 could be fully recovered from plasma and urine. The normal reference range for MT-1 was 32 +/- 16 ng/ml in plasma and 10 +/- 6 ng MT-1 per micromole of creatinine in random samples of urine. No significant differences were found between the values for males and females. The concentration of MT-1 was greatly increased between 24 and 48 hours after surgery, indicating that the protein behaves like an acute phase reactant in human subjects.

Topics: Acute-Phase Proteins; Acute-Phase Reaction; Adult; Aged; Aged, 80 and over; Animals; Antibodies; Antibody Specificity; Chromatography, Gel; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Female; Humans; Liver; Male; Metallothionein; Middle Aged; Rabbits; Sensitivity and Specificity

An acute phase response, a group of adaptations to some types of stress, blocks injury in rodents due to hepatotoxins and agents generating arthritis-like inflammation. In contrast, this study found no protection against adriamycin-induced acute cardiotoxicity in rats. The acute phase response was initiated by turpentine-stimulated leg inflammation. Injury was assessed by survival, macroscopic signs of injury, and heart lipid peroxidation. Acute phase response produced the expected rises in the stress-responsive proteins: serum ceruloplasmin and liver metallothionein. However, cardiac metallothionein was unaffected. These results suggest that an acute phase response will not necessarily protect tissues where levels of stress-induced proteins are not raised.

Topics: Acute-Phase Reaction; Animals; Ceruloplasmin; Doxorubicin; Heart; Inflammation; Lipid Peroxidation; Liver; Male; Metallothionein; Myocardium; Rats; Rats, Sprague-Dawley; Turpentine

Topics: Acute-Phase Reaction; Animals; Antibodies; Gene Expression; Horses; Humans; Metallothionein; Metals; Mice

Low zinc (Zn) intake could be expected to compromise resistance to oxidative stress, even when accompanied by a normally protective acute phase response pretreatment. Mildly Zn deficient rats showed very high degrees of CCl4-induced hepatic cell membrane injury as assessed by serum sorbitol dehydrogenase activities. Rats pair-fed adequate Zn also showed above normal degrees of injury, but much less than rats fed low Zn. An acute phase response, elicited by leg inflammation, strongly protected rats consuming adequate Zn, either ad libitum or pair-fed, against the CCl4-induced rise in sorbitol dehydrogenase. However, the effect was partially absent in rats fed low Zn. Zinc intake had no effect on CCl4-produced microsomal injury, assessed by glucose-6-phosphatase activities. Rats fed low Zn showed normal hepatic levels of metallothionein, a Zn protein with proposed antioxidant functions, but did not show the rise in metallothionein levels normally associated with acute phase response. In summary, mild Zn deficiency caused poor resistance to CCl4-induced plasma membrane injury and partially negated acute phase response protective effects. Metallothionein was not involved in the former effect, but may have contributed to the latter.

Topics: Acute-Phase Reaction; Animals; Carbon Tetrachloride; Cell Membrane; Chemical and Drug Induced Liver Injury; Glucose-6-Phosphatase; L-Iditol 2-Dehydrogenase; Liver; Male; Metallothionein; Rats; Rats, Sprague-Dawley; Zinc

Induction of hepatic metallothionein (MT) synthesis by several nonmetallic compounds and its relationship to an acute-phase response in inflammation were studied in mice. Subcutaneous injections of menadione, paraquat, carbon tetrachloride (CCl4), and several organic solvents caused an increase of hepatic MT concentration. This MT contained only zinc. Menadione and n-hexane caused the greatest accumulation of hepatic MT among these nonmetallic compounds (about 13-fold). The concentration of Zn was significantly decreased in plasma in contrast to liver after an injection of these nonmetallic compounds. When 65ZnCl2 was injected iv after these injections, uptake of 65Zn to the liver was increased. This effect was not observed after treatment with cycloheximide. The association with inflammation of this induction of MT accumulation was examined by determination of acute-phase proteins. The concentration of fibrinogen in the plasma was significantly increased following injection of those nonmetallic compounds which caused marked hepatic MT accumulation. An injection of 1 N NaOH, 1 N HCl, turpentine oil, or endotoxin caused a significant increase in the plasma concentration of fibrinogen and in the hepatic MT concentration. Injections of n-hexane as well as turpentine oil significantly increased hepatic MT concentration and plasma concentration of fibrinogen and ceruloplasmin with time. The concentration of fibrinogen was significantly correlated (r = 0.789) with the concentration of hepatic MT. Neither adrenalectomy nor pretreatment with dexamethasone prevented hepatic MT accumulation caused by these compounds. These results indicate that induction of hepatic MT synthesis by these nonmetallic compounds is associated with an acute-phase response in inflammation and is independent of glucocorticoids.

Topics: Acute-Phase Proteins; Acute-Phase Reaction; Animals; Fibrinogen; Glucocorticoids; Hexanes; Hydrochloric Acid; Inflammation; Lipopolysaccharides; Liver; Male; Metallothionein; Mice; Mice, Inbred Strains; Sodium Hydroxide; Turpentine; Vitamin K; Zinc

Interleukin 1 (IL 1) production is stimulated by infection, cellular injury, and inflammation. This cytokine directs a wide spectrum of host responses. Human interleukin 1 alpha (IL 1 alpha) was used to examine the time course of effects on zinc metabolism as part of the acute phase response. IL 1 produced a transient depression in the serum zinc concentration and increased serum ceruloplasmin. Metallothionein levels were increased in liver 14-fold after IL 1. Increased expression of metallothionein-1 and -2 genes following IL 1 were observed in liver, bone marrow, and thymus. Pulse-labeling experiments with i.v.-administered 65Zn showed that IL 1 drastically altered zinc distribution kinetics among tissues. More 65Zn was taken up (and/or retained) by the liver, bone marrow, and thymus 6 h after IL 1, whereas correspondingly less 65Zn was found in bone, skin, and intestine. Uptake by other tissues was not affected by IL 1. Chromatography of cytosol from tissues with increased 65Zn uptake suggests the IL 1-induced redistribution may be driven by enhanced metallothionein synthesis. Collectively, the results show that IL 1 regulates zinc metabolism and may direct its preferential, tissue-specific distribution via elevated metallothionein-1 and -2 gene expression.

Topics: Acute-Phase Reaction; Animals; Blotting, Northern; Bone Marrow; Ceruloplasmin; Chromatography, Gel; Chromatography, Ion Exchange; Gene Expression Regulation; Interleukin-1; Liver; Male; Metallothionein; Rats; Recombinant Proteins; RNA; RNA, Messenger; Serum Albumin; Thymus Gland; Tissue Distribution; Zinc

Acute phase changes in trace mineral metabolism were examined in turkey embryos. An endotoxin injection resulted in increased concentrations of serum copper and liver zinc and decreased concentrations of serum zinc in embryos incubated either in ovo or ex ovo. Changes in zinc and copper metabolism occurred when endotoxin either was injected intramuscularly, into the amnionic fluid, or administered onto the chorioallantoic membrane. Unlike poults, embryos did not respond to an inflammatory challenge with decreased serum iron concentrations. Acute phase changes in embryo serum zinc and copper as well as liver zinc concentrations were similar to those in poults. Increased liver zinc concentrations were associated with increased zinc in metallothionein (MT). An injection of a crude interleukin 1 preparation into embryos resulted in similar increases in hepatic zinc and MT concentrations as an endotoxin injection, suggesting a role for this cytokine in mediating the acute phase changes in embryonic zinc metabolism.

Topics: Acute-Phase Reaction; Age Factors; Animals; Bacterial Toxins; Copper; Endotoxins; Escherichia coli; Inflammation; Interleukin-1; Iron; Liver; Metallothionein; Turkeys; Zinc