Selective induction of IL-6 by aluminum-induced oxidative stress can be prevented by selenium

In this study the acute toxic effects of aluminum (Al) on mice have been investigated, including the interactions of Al and selenium (Se). Focus was put on the systemic effects of (co)exposure to Al and Se as a reflection of the redox status in the liver, kidney and brain.Short-term exposure (16 h) to Al resulted in an increase in the systemic inflammation parameters IL-6 and PAI-1, whereas serum levels of TNF-α remained unaffected. The different response pattern of IL-6 and TNF-α probably indicates an increased intracellular oxidative stress and altered redox status in the liver, because the selective increase in IL-6 serves as a protective intrahepatocellular process driven by oxidative stress. The intracellular glutathione concentration GSH_tot decreased significantly upon Al exposure. Both the increase in IL-6 and decrease in glutathione status could be prevented by co-exposure to Se, but not the increase in PAI-1. The redox status of the kidney and brain was not markedly affected.Therefore it was concluded that short-term exposure to Al causes adverse effects on the intracellular oxidative stress processes in the liver, as reflected by the selective increase in the IL-6 concentration. This process can be restored by co-administration of the trace element Se as a part of the glutathione redox system.     

Metallothionein induction by nonsteroidal antiinflammatory drugs

In the present study we report on the effects of commonly used nonsteroidal antiinflammatory drugs on metallothionein (MT) and MT-I mRNA levels. A single dose of chloroquine (100 mg/kg), diclofenac (100 mg/kg), indomethacin (10 mg/kg), or piroxicam (100 mg/kg) was administered ip to C57B1 mice. After 18 h, MT levels were determined with a Cd-saturation radioassay. MT-I mRNA levels were measured by Northern Blot analyses using a probe containing the mouse MT-I gene. All drugs tested caused an increase in the MT content of the liver but not of the kidneys and lung. The lowest and highest effects were observed with chloroquine (8 times the control value) and diclofenac (18 times), respectively. In accordance with the stimulation of MT synthesis, increased accumulation of hepatic MT-I mRNA could be demonstrated. These results indicate that elevated MT levels may contribute to the effectiveness of nonsteroidal antiinflammatory drugs in the treatment of rheumatoid arthritis (RA).     

Upregulation of IL-6 mRNA by IL-6 in skeletal muscle cells: role of IL-6 mRNA stabilization and Ca2+-dependent mechanisms

Skeletal muscle cells have been established as significant producers of IL-6 during exercise. This IL-6 production is discussed as one possible mediator of the beneficial effects of physical activity on glucose and fatty acid metabolism. IL-6 itself could be the exercise-related factor that upregulates and maintains its own production. We investigated this hypothesis and the underlying molecular mechanism in cultured C₂C₁₂ cells. IL-6 led to a rapid and prolonged increase in IL-6 mRNA, which was also found in human myotubes. Because IL-6 has been shown to activate AMP-activated kinase (AMPK), we studied whether, in turn, activated AMPK induces IL-6 expression. Pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide-1--4-ribofuranoside upregulated IL-6 mRNA expression, which was blocked by knockdown of AMPK ₁ and ₂ using small, interfering RNA (siRNA) oligonucleotides. However, the effect of IL-6 was shown to be independent of AMPK, since the siRNA approach silencing the AMPK -subunits did not reduce the upregulation of IL-6 induced by IL-6 stimulation. The self-stimulatory effect of IL-6 partly involves a Ca²⁺-dependent pathway: IL-6 increased intracellular Ca²⁺, and intracellular blockade of Ca²⁺ with a Ca²⁺ chelator reduced the IL-6-mediated increase in IL-6 mRNA levels. Moreover, inhibition of Ca²⁺/calmodulin-dependent kinase kinase with STO-609 or the siRNA approach decreased IL-6 mRNA levels of control and IL-6-stimulated cells. A major, STO-609-independent mechanism is the IL-6-mediated stabilization of its mRNA. The data suggest that IL-6 could act as autocrine factor upregulating its mRNA levels, thereby supporting its function as an exercise-activated factor in skeletal muscle cells. 5-aminoimidazole-4-carboxamide-1--4-ribofuranoside; AMP-activated kinase; STO-609; calcium/calmodulin-dependent kinase kinase; C₂C₁₂ cells     

IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.     

TGF-β is necessary for induction of IL-23R and Th17 differentiation by IL-6 and IL-23

TGF-β and IL-6 induce Th17 differentiation, and IL-23 is required for expansion and maintenance of Th17 cells. Recently, it was shown that IL-6 up-regulates IL-23R mRNA in naive CD4⁺ T cells and therefore IL-6 and IL-23 synergistically promote Th17 differentiation. However, the molecular mechanism whereby IL-6 and IL-23 induce Th17 differentiation and the relevance to TGF-β remain unknown. Here, we found that IL-6 up-regulated IL-23R mRNA expression, and IL-6 and IL-23 synergistically augmented its protein expression. The combination induced Th17 differentiation, and TGF-β1 further enhanced it. IL-6 augmented endogenous TGF-β1 mRNA expression, whereas the amount of TGF-β produced was not enough to induce Th17 differentiation by IL-6 alone. However, unexpectedly, the up-regulation of IL-23R and induction of Th17 differentiation by IL-6 and IL-23 were almost completely inhibited by anti-TGF-β. These results suggest that the induction of IL-23R and Th17 differentiation by IL-6 and IL-23 is mediated through endogenously produced TGF-β.     

Inhibition of IL-6 and IL-10 signaling and Stat activation by inflammatory and stress pathways

The development and resolution of an inflammatory process are regulated by a complex interplay among cytokines that have pro- and anti-inflammatory effects. Effective and sustained action of a proinflammatory cytokine depends on synergy with other inflammatory cytokines and antagonism of opposing cytokines that are often highly expressed at inflammatory sites. We analyzed the effects of the inflammatory and stress agents, IL-1, TNF-alpha, LPS, sorbitol, and H(2)O(2), on signaling by IL-6 and IL-10, pleiotropic cytokines that activate the Jak-Stat signaling pathway and have both pro- and anti-inflammatory actions. IL-1, TNF-alpha, and LPS blocked the activation of Stat DNA binding and tyrosine phosphorylation by IL-6 and IL-10, but not by IFN-gamma, in primary macrophages. Inhibition of Stat activation correlated with inhibition of expression of IL-6-inducible genes. The inhibition was rapid and independent of de novo gene induction and occurred when the expression of suppressor of cytokine synthesis-3 was blocked. Inhibition of IL-6 signaling was mediated by the p38 subfamily of stress-activated protein kinases. Jak1 was inhibited at the level of tyrosine phosphorylation, indicating that inhibition occurred at least in part upstream of Stats in the Jak-Stat pathway. Experiments using Stat3 mutated at serine 727 and using truncated IL-6Rs suggested that the target of inhibition is contained within the membrane-proximal region of the cytoplasmic domain of the gp130 subunit of the IL-6 receptor and is different from the SH2 domain-containing protein-tyrosine phosphatase/suppressor of cytokine synthesis-3 docking site. These results identify a new level at which IL-1 and TNF-alpha modulate signaling by pleiotropic cytokines such as IL-6 and IL-10 and provide a molecular basis for the previously described antagonism of certain IL-6 actions by IL-1.     

Cocaine fatalities increased by restraint stress

Laboratory rats injected daily with a moderate dose of cocaine hydrochloride (30 mg/kg, i.p.) showed increased fatalities when cocaine injections were followed by 30 min of restraint stress. The 5-day mortality rate was 58% for the cocaine-plus-stress group, while 17% of the animals receiving cocaine without restraint stress died. This finding suggests that stress can augment the toxic effect of cocaine and that minimizing stress may be an important consideration in the clinical management of cocaine overdose.     

Metallothionein mRNA and protein induction by cadmium in peripheral-blood leucocytes.

To help to evaluate the role of metallothionein (MT) in peripheral-blood leucocytes, we examined MT protein and mRNA levels in these cells before and after exposure to CdCl2 in culture. Protein was assayed by 109Cd2+ binding, and RNA by dot-blot hybridization. MT was induced in both lymphocytes and adherent monocytes about 10-fold with a 12 h exposure to 10 microM-CdCl2, but absolute levels were 3-fold higher in monocytes: 57 x 10(5) (+Cd2+) versus 6 x 10(5) (-Cd2+) molecules/cell for monocytes; 18 x 10(5) (+Cd2+) versus 2 x 10(5) (-Cd2+) for lymphocytes. Polymorphonuclear cells expressed relatively little MT (0.6 x 10(5) molecules/cell), and this did not change with phorbol ester stimulation or exposure to Cd2+, arguing against a direct protective role for MT in activated neutrophils. MT mRNA levels corresponded qualitatively to expression of protein in these cells. Our data provide quantitative comparisons of leucocyte MT expression and regulation in the human population. Variation in both basal and induced MT mRNA levels reflects environmental or experimental (intra-individual) and possibly genetic (inter-individual) differences.     

Metallothionein induction in human peripheral blood lymphocytes by heavy metals

Human peripheral blood lymphocytes have the capacity to produce metallothioneins (MTs) as a protective response to cadmium exposure. To define the range of metal species inducing lymphocyte MTs, cellular proteins synthesized after exposure to each of 11 heavy metals were analyzed by gel electrophoresis. Toxic metals such as cadmium, mercury and silver were found to induce thioneins (apoproteins of MTs) at relatively low concentrations (maximum at approximately 10 microM), whereas less toxic metals such as zinc, copper and nickel were inductive at relatively high concentrations (maximum at approximately 200 microM). Tin, lead, iron, cobalt, and manganese did not induce thioneins. The heavy metal specificity of MT induction in the lymphocyte resembles that in the liver, and the regulatory mechanism of MT production seems to be similar in both of these tissues. In the cells exposed to highly toxic metals such as cadmium and mercury, expression of cytotoxicity (represented by decline of cysteine uptake) was remarkable at the metal concentrations higher than those saturating thionein induction, supporting the protective role of MTs against heavy metals.     

CD38 ligation plays a direct role in the induction of IL-1beta,IL-6, and IL-10 secretion in resting human monocytes

CD38 signaling, either induced by ligation with specific agonistic monoclonal antibody (mAb) or after interaction with CD31, its cognate counter-receptor, is involved in release of IL-1beta, IL-6, and IL-10 cytokines in resting human monocytes. CD38 ligation by the F(ab')(2) IB4 mAb did not induce signals relevant for cytokine secretion and the block of the Fcgamma receptor I (FcgammaRI) by anti-CD64 or FcgammaRII by anti-CD32 mAb did not inhibit CD38-mediated IL-1beta release. Dimerization or multimerization of the CD38 molecule by: (i) cross-linking of the receptor ligated by F(ab')(2) or by (ii) increasing CD38 expression by treating monocytes with IFNgamma were able to restore the truncated CD38-mediated signals involved in cytokine secretion. These data indicate that CD38 receptor-mediated signals operate directly suggesting a Fcgamma receptorial surface molecule independent activation pathway. The key element for the receptor mediated signaling is represented by surface density of CD38 on resting monocytes.     

Dual role of the IL-12/IFN-gamma axis in the development of autoimmune myocarditis: induction by IL-12 and protection by IFN-gamma.

IL-12 and IFN-gamma positively regulate each other and type 1 inflammatory responses, which are believed to cause tissue damage in autoimmune diseases. We investigated the role of the IL-12/IFN-gamma (Th1) axis in the development of autoimmune myocarditis. IL-12p40-deficient mice on a susceptible background resisted myocarditis. In the absence of IL-12, autospecific CD4(+) T cells proliferated poorly and showed increased Th2 cytokine responses. However, IFN-gamma-deficient mice developed fatal autoimmune disease, and blockade of IL-4R signaling did not confer susceptibility to myocarditis in IL-12p40-deficient mice, demonstrating that IL-12 triggers autoimmunity by a mechanism independent of the effector cytokines IFN-gamma and IL-4. In conclusion, our results suggest that the IL-12/IFN-gamma axis is a double-edged sword for the development of autoimmune myocarditis. Although IL-12 mediates disease by induction/expansion of Th1-type cells, IFN-gamma production from these cells limits disease progression.     

In vivo induction of IL-6 by administration of exogenous cytokines and detection of de novo serum levels of IL-6 in tumor-bearing mice

We investigated the capacity of several recombinant cytokines to induce IL-6 in vivo in both normal and tumor-bearing (TB) mice. Intravenous administration of human rhTNF-alpha, rhIL-1, rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma were all capable of inducing circulating IL-6. rhTNF-alpha administration caused the greatest induction of IL-6. TB animals consistently produced more IL-6 in response to rhTNF-alpha than did normal mice (2 h after 4 micrograms rhTNF-alpha, TB = 24,100 HGF U/ml, non-TB = 3600 HGF U/ml of IL-6). A single daily i.v. dose of rhTNF-alpha (4 micrograms/mouse/day) for 5 days led to decreased IL-6 induction in TB animals by day 3 of treatment (peak levels of IL-6, day 1 = 72,800 HGF U/ml, day 3 = 23,400 HGF U/ml, day 5 = 26,400 HGF U/ml). rhIL-1 administration also resulted in considerable IL-6 production, although peak values were less than those resulting from administration of rhTNF-alpha. Administration of rhIL-1 induced similar IL-6 levels (TB = 10,025 and non-TB = 10,600 HGF U/ml) in TB and normal mice. Single high doses of rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma induced lower but consistent levels of circulating IL-6 in mice with and without tumor. In addition, the sera of untreated TB mice contained levels of IL-6 which paralleled the extent of tumor burden (serum IL-6 in day 30 MCA 106 TB mice = 420 HGF U/ml). The detection of de novo IL-6 was also confirmed in animals bearing tumors of different histologies (the MCA 102 sarcoma, MCA 38 adenocarcinoma, and B16 melanoma). At no time was IL-6 measurable in the sera of untreated normal mice. The identification of IL-6 was verified by neutralization studies using specific antimurine IL-6 antibody. Although the exact role of IL-6 in TB animals remains to be elucidated, its known pleotrophic immune and metabolic effects may be important in the host response to malignancy.     

Effect of restraint stress on nociceptive responses in rats: role of the histaminergic system

Stress induced analgesia (SIA) is well known, but the reverse phenomenon, hyperalgesia is poorly documented. This study investigated the role of the histaminergic system in restraint stress hyperalgesia in rats, using thermal stimulation method (hot plate and tail flick tests). Paw licking and tail withdrawal latencies were taken before and after restraint for about one hour. Significant decreases were obtained in these latencies after the restraint in both tests. Administration of H1 and H2 receptor blockers, chlorpheniramine and cimetidine respectively 30 mins before the restraint still resulted in significant reductions in these latencies, connoting the persistence of hyperalgesia, showing that histamine H1 and H2 receptors did not participate in the mechanism of restraint stress hyperalgesia. We therefore suggest a histaminergic independent mechanism for restraint stress induced hyperalgesia.