Matjaz Humar

Volatile anesthetics induce caspase-dependent, mitochondria-mediated apoptosis in human T lymphocytes in vitro.

Background:Volatile anesthetics modulate lymphocyte function during surgery, and this compromises postoperative immune competence. The current work was undertaken to examine whether volatile anesthetics induce apoptosis in human T lymphocytes and what apoptotic signaling pathway might be used. Methods:Effects of sevoflurane, isoflurane, and desflurane were studied in primary human CD3+ T lymphocytes and Jurkat T cells in vitro. Apoptosis and mitochondrial membrane potential were assessed using flow cytometry after green fluorescent protein-annexin V and DiOC6-fluorochrome staining. Activity and proteolytic processing of caspase 3 was measured by cleaving of the fluorogenic effector caspase substrate Ac-DEVD-AMC and by anti–caspase-3 Western blotting. Release of mitochondrial cytochrome c was studied after cell fractionation using anti–cytochrome c Western blotting and enzyme-linked immunosorbent assays. Results:Sevoflurane and isoflurane induced apoptosis in human T lymphocytes in a dose-dependent manner. By contrast, desflurane did not exert any proapoptotic effects. The apoptotic signaling pathway used by sevoflurane involved disruption of the mitochondrial membrane potential and release of cytochrome c from mitochondria to the cytosol. In addition, the authors observed a proteolytic cleavage of the inactive p32 procaspase 3 to the active p17 fragment, increased caspase-3–like activity, and cleavage of the caspase-3 substrate poly-ADP-ribose-polymerase. Sevoflurane-induced apoptosis was blocked by the general caspase inhibitor Z-VAD.fmk. Death signaling was not mediated via the Fas/CD95 receptor pathway because neither anti-Fas/CD95 receptor antagonism nor FADD deficiency or caspase-8 deficiency were able to attenuate sevoflurane-mediated apoptosis. Conclusion:Sevoflurane and isoflurane induce apoptosis in T lymphocytes via increased mitochondrial membrane permeability and caspase-3 activation, but independently of death receptor signaling.

Heme oxygenase-1 induction by the clinically used anesthetic isoflurane protects rat livers from ischemia/reperfusion injury.

Objective:It was the aim of this study to characterize the influence of isoflurane-induced heme oxygenase-1 (HO-1) expression on hepatocellular integrity after ischemia and reperfusion. Summary Background Data:Abundant experimental data characterize HO-1 as one of the most powerful inducible enzymes that contribute to the protection of the liver and other organs after harmful stimuli. Therapeutic strategies aimed at utilizing the protective effects of HO-1 are hampered by the fact that most pharmacological inducers of this enzyme perturb organ function by themselves and are not available for use in patients because of their toxicity and undesirable or unknown side effects. Methods:Rats were pretreated with isoflurane before induction of partial hepatic ischemia (1 hour) and reperfusion (1 hour). At the end of each experiment, blood and liver tissue were obtained for molecular biologic, histologic, and immunohistochemical analyses. Results:Isoflurane pretreatment increased hepatic HO-1 mRNA, HO-1 protein, HO enzyme activity, and decreased plasma levels of AST, ALT, and α-GST. Histologic analysis of livers obtained from isoflurane-pretreated rats showed a reduction of necrotic areas, particularly in the perivenular region, the predominant site of isoflurane-induced HO-1 expression. In addition, sinusoidal congestion that could otherwise be observed after ischemia/reperfusion was inhibited by the anesthetic. Furthermore, isoflurane augmented hepatic microvascular blood flow and lowered the malondialdehyde content within the liver compared with control animals. Administration of tin protoporphyrin IX inhibited HO activity and abolished the isoflurane-induced protective effects. Conclusions:This study provides first evidence that pretreatment with the nontoxic and clinically approved anesthetic isoflurane induces hepatic HO-1 expression, and thereby protects rat livers from ischemia/reperfusion injury.

Thiopental Inhibits the Activation of Nuclear Factor κB

Background Thiopental is frequently used for the treatment of intracranial hypertension after severe head injury. Its long-term administration increases the incidence of nosocomial infections, which contributes to the high mortality rate of these patients. However, the mechanism of its immunosuppressing effect remains unknown. Methods The effect of thiopental (200–1000 &mgr;g/ml) on the activation of the nuclear transcription factor &kgr;B (NF-&kgr;B; electrophoretic mobility shift assays), on NF-&kgr;B–driven reporter gene activity (transient transfection assays), on the expression of NF-&kgr;B target genes (enzyme-linked immunoassays), on T-cell activation (flow cytometric analyses of CD69 expression), and on the content of the NF-&kgr;B inhibitor I&kgr;B-&agr; (Western blotting) was studied in human T lymphocytes in vitro. Results Thiopental inhibited the activation of the transcription factor NF-&kgr;B but did not alter the activity of the cyclic adenosine monophosphate response element binding protein. Other barbiturates (methohexital), anesthetics (etomidate, propofol, ketamine), or opioids (fentanyl, morphine) did not affect NF-&kgr;B activation. Thiopental-mediated suppression of NF-&kgr;B could be observed in Jurkat cells and in primary CD3+ lymphocytes from healthy volunteers, was time- and concentration-dependent, occurred at concentrations that are clinically achieved, and persisted for hours after the incubation. It was associated with an inhibition of NF-&kgr;B–driven reporter gene activity, of the expression of interleukin-2, -6, and -8, and interferon &ggr;, and of the activation of CD3+ lymphocytes. Suppression of NF-&kgr;B appeared to involve reduced degradation of I&kgr;B-&agr;. Conclusion The results demonstrate that thiopental inhibits the activation of NF-&kgr;B and may thus provide a molecular mechanism for some of the immunosuppressing effects associated with thiopental therapy.

Thiopental Inhibits Tumor Necrosis Factor α–induced Activation of Nuclear Factor κB through Suppression of IκB Kinase Activity

Background Thiopental is frequently used for the treatment of intracranial hypertension after severe head injury and is associated with immunosuppressive effects. The authors have recently reported that thiopental inhibits activation of nuclear factor (NF) &kgr;B, a transcription factor implicated in the expression of many inflammatory genes. Thus, it was the aim of the current study to examine the molecular mechanism of this inhibitory effect. Methods The authors tested &ggr;-aminobutyric acid (GABA), the GABAA antagonist bicuculline, and the GABAB antagonist dichlorophenyl-methyl-amino-propyl-diethoxymethyl-phosphinic acid (CGP 52432) in combination with thiopental for their influence on the activation of NF-&kgr;B. In addition, they investigated the direct effect of thiopental on activated NF-&kgr;B DNA binding activity. These experiments were conducted in Jurkat T lymphocytes using electrophoretic mobility shift assays. The presence of the phosphorylated and dephosphorylated NF-&kgr;B inhibitor I&kgr;B&agr; (Western blotting) and I&kgr;B kinase activity were studied in Jurkat T cells and human CD3+ T lymphocytes. In addition, the authors tested the effect of the structural barbiturate analog pairs thiopental–pentobarbital and thiamylal–secobarbital and of thiopental in combination with the thio-group containing chemical dithiothreitol on the activation of NF-&kgr;B. Results GABA did not inhibit NF-&kgr;B activation, and the GABAA and GABAB antagonists bicuculline and CGP did not diminish the thiopental-mediated inhibitory effect on NF-&kgr;B activation. Thiopental did not inhibit activated NF-&kgr;B directly in a cell-free system. The phosphorylation of I&kgr;B&agr; was prevented after incubation with 1,000 &mgr;g/ml thiopental. The same concentration of thiopental also inhibited I&kgr;B kinase activity in tumor necrosis factor–stimulated Jurkat T cells and human CD3+ T lymphocytes (60% suppression, P < 0.05 vs. tumor necrosis factor &agr; alone). Thiobarbiturates (4 × 10−3 m) inhibited NF-&kgr;B activity, whereas equimolar concentrations of the structural oxyanalogs did not. Preincubation of thiopental with dithiothreitol diminished the inhibitory effect. Conclusion Thiopental-mediated inhibition of NF-&kgr;B activation is due to the suppression of I&kgr;B kinase activity and depends at least in part on the thio-group of the barbiturate molecule.

Mechanism of hepatic heme oxygenase-1 induction by isoflurane

Background:The heme oxygenase pathway represents a major cell and organ protective system in the liver. The authors recently showed that isoflurane and sevoflurane up-regulate the inducible isoform heme oxygenase 1 (HO-1). Because the activating cascade remained unclear, it was the aim of this study to identify the underlying mechanism of this effect. Methods:Rats were anesthetized with pentobarbital intravenously or with isoflurane per inhalation (2.3 vol%). Kupffer cell function was inhibited by dexamethasone or gadolinium chloride. Nitric oxide synthases were inhibited by either N&ohgr;-nitro-l-arginine methyl ester or S-methyl thiourea. N-Acetyl-cysteine served as an antioxidant, and diethyldithiocarbamate served as an inhibitor of cytochrome P450 2E1. Protein kinase C and phospholipase A2 were inhibited by chelerythrine or quinacrine, respectively. HO-1 was analyzed in liver tissue by Northern blot, Western blot, immunostaining, and enzymatic activity assay. Results:In contrast to pentobarbital, isoflurane induced HO-1 after 4–6 h in hepatocytes in the pericentral region of the liver. The induction was prevented in the presence of dexamethasone (P < 0.05) and gadolinium chloride (P < 0.05). Inhibition of nitric oxide synthases or reactive oxygen intermediates did not affect isoflurane-mediated HO-1 up-regulation. In contrast, chelerythrine (P < 0.05) and quinacrine (P < 0.05) resulted in a blockade of HO-1 induction. Conclusion:The up-regulation of HO-1 by isoflurane in the liver is restricted to parenchymal cells and depends on Kupffer cell function. The induction is independent of nitric oxide or reactive oxygen species but does involve protein kinase C and phospholipase A2.

Differential Effects of Volatile Anesthetics on Hepatic Heme Oxygenase–1 Expression in the Rat

THE microsomal enzyme heme oxygenase (HO; EC 1.14.99.3) catalyzes the oxidation of heme to biliverdinIXa, iron, and carbon monoxide. So far, three isoforms of this enzyme have been cloned. While HO-2 and HO-3 are constitutively expressed, HO-1 is highly inducible in response to a variety of stimuli and has been identified as the major 32-kd heat shock (stress) protein (HSP) 32. Heme oxygenase–1 is essential for the function of the normal liver and plays a major protective role in the stress-exposed liver. It exerts antioxidant properties by cleavage of the prooxidant heme and by the production of biliverdin, which is subsequently reduced to the antioxidant bilirubin. Furthermore, its product carbon monoxide serves to maintain liver perfusion and has potent antiinflammatory effects. Many studies have shown differential effects of volatile anesthetics on the perfusion, function, and integrity of the liver. Moreover, preliminary evidence suggests that volatile anesthetics may interfere with stress gene expression and function. We therefore aimed to determine whether volatile anesthetics affect hepatic HO-1 gene expression.

Heme Oxygenase-1 Inhibits the Proliferation of Pancreatic Stellate Cells by Repression of the Extracellular Signal-Regulated Kinase1/2 Pathway

Activation of pancreatic stellate cells (PSCs) is the key process in the development of pancreatic fibrosis, a common feature of chronic pancreatitis and pancreatic cancer. In recent studies, curcumin has been shown to inhibit PSC proliferation via an extracellular signal-regulated kinase (ERK)1/2-dependent mechanism. In addition, curcumin is a potent inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1) in other cell types. Therefore, the aims of this study were to 1) characterize the effect of curcumin on HO-1 gene expression in PSCs, 2) explore whether HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation, and 3) clarify the involvement of the mitogen-activated protein kinase (MAPK) family in this context. Cultured rat PSCs were incubated with curcumin and assessed for HO-1 up-regulation by Northern blot analysis, immunoblotting, and activity assays. The effect of HO-1 on platelet-derived growth factor (PDGF)-induced PSC proliferation and MAPK activation was determined by immunoblotting, cell proliferation assays, and cell count analyses. Curcumin induced HO-1 gene expression in PSCs in a time- and dose-dependent manner and inhibited PDGF-mediated ERK1/2 phosphorylation and PSC proliferation. These effects were blocked by treatment of PSCs with tin protoporphyrin IX, an HO inhibitor, or transfection of HO-1 small interfering RNA. Our data provide evidence that HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation. Therefore, therapeutic up-regulation of HO-1 could represent a mode for inhibition of PSC proliferation and thus may provide a novel strategy in the prevention of pancreatic fibrosis.

Sevoflurane inhibits phorbol-myristate-acetate-induced activator protein-1 activation in human T lymphocytes in vitro: Potential role of the p38-stress kinase pathway

Background: Modulation of immune defense mechanisms by volatile anesthetics during general anesthesia may compromise postoperative immune competence and healing reactions and affect the infection rate and the rate of tumor metastases disseminated during surgery. Several mechanisms have been suggested to account for these effects. The current study was undertaken to examine the molecular mechanisms underlying these observations. Methods: Effects of sevoflurane, isoflurane, and desflurane were studied in vitro in primary human CD3+ T-lymphocytes. DNA-binding activity of the transcription factor activator protein-1 (AP-1) was assessed using an electrophoretic mobility shift assay. Phorbol-myristate-acetate-dependent effects of sevoflurane on the phosphorylation of the mitogen-activated protein kinases were studied using Western blots, the trans-activating potency of AP-1 was determined using reporter gene assays, and the cytokine release was measured using enzyme-linked immunosorbent assays. Results: Sevoflurane inhibited activation of the transcription factor AP-1. This effect was specific, as the activity of nuclear factor &kgr;B, nuclear factor of activated T cells, and specific protein-1 was not altered and several other volatile anesthetics studied did not affect AP-1 activation. Sevoflurane-mediated suppression of AP-1 could be observed in primary CD3+ lymphocytes from healthy volunteers, was time-dependent and concentration-dependent, and occurred at concentrations that are clinically achieved. It resulted in an inhibition of AP-1-driven reporter gene activity and of the expression of the AP-1 target gene interleukin-3. Suppression of AP-1 was associated with altered phosphorylation of p38 mitogen-activated protein kinases. Conclusion: The data demonstrate that sevoflurane is a specific inhibitor of AP-1 and may thus provide a molecular mechanism for the antiinflammatory effects associated with sevoflurane administration.

Thiopental protects human T lymphocytes from apoptosis in vitro via the expression of heat shock protein 70

Barbiturates, which are used for the treatment of intracranial hypertension after severe head injury, have been associated with anti-inflammatory side effects. Although all barbiturates inhibit T-cell function, only thiobarbiturates markedly reduce the activation of the transcription factor nuclear factor-κB (NF-κB). Various pharmacologic inhibitors of the NF-κB pathway are concomitant nonthermal inducers of the heat shock response (HSR), a cellular defense system that is associated with protection of cells and organs. We hypothesize that thiopental mediates cytoprotection by inducing the HSR. Human CD3+ T lymphocytes were incubated with thiopental, pentobarbital, etomidate, ketamine, midazolam, or propofol. Human Jurkat T cells were transfected with small interfering RNA (siRNA) targeting heat 70-kDa shock protein (hsp 70) before thiopental incubation. Apoptosis was induced by staurosporine. DNA binding activity of HSF-1 was analyzed by electrophoretic mobility shift assay; mRNA expression of hsp27, -32, -70, and -90 was analyzed by Northern blot, and protein expression of hsp70 was analyzed by Western blot and flow cytometry after fluorescein isothiocyanate (FITC)-hsp70-antibody staining. Apoptosis was assessed by flow cytometry after annexin V-FITC or annexin V-phycoerythrin staining. Activity of caspase-3 was measured by fluorogenic caspase activity assay. Thiopental induced hsp27, -70, and -90 but not hsp32 mRNA expression as well as hsp70 protein expression. Thiopental dose-dependently activated the DNA binding activity of HSF-1, whereas other substances investigated had no effect. In addition, pretreatment with thiopental significantly attenuated staurosporine-induced apoptosis and caspase-like activity. Transfection with hsp70-siRNA before thiopental treatment reduced this attenuation. Thiopental specifically and differentially induces a heat shock response, and it mediates cytoprotection via the expression of hsp70 in human T lymphocytes.

Dobutamine Inhibits Phorbol-Myristate-Acetate-Induced Activation of Nuclear Factor-??B in Human T Lymphocytes In Vitro

Adrenergic drugs are often used for hemodynamic support of cardiac output and vasomotor tone in critically ill patients. Recent evidence shows that the administration of these vasoactive drugs may affect cytokine release and could influence the inflammatory response. However, the mechanism of this immunomodulatory effect remains unknown. The nuclear transcription factor-&kgr;B (NF-&kgr;B) regulates the expression of many cytokines and plays a central role in the immune response. Therefore, we examined the effects of various adrenergic drugs (dobutamine, xamoterol, clenbuterol, epinephrine, norepinephrine, and phenylephrine) on the activation of NF-&kgr;B, on the NF-&kgr;B-driven reporter gene activity, and on the expression of the NF-&kgr;B target gene interleukin (IL)-8. In addition, we quantified the amount of the NF-&kgr;B inhibitors I&kgr;B&agr; and IL-10. Here we report that dobutamine inhibited the activation of NF-&kgr;B in primary human CD3+ T lymphocytes. Suppression of NF-&kgr;B involved the stabilization of its inhibitor, I&kgr;B&agr;. The effect appears to be &bgr;2-receptor specific, because &bgr;1-adrenergic and &agr;-adrenergic substances (i.e., xamoterol, epinephrine, norepinephrine, and phenylephrine) did not affect NF-&kgr;B activation and because dobutamine-mediated inhibition of NF-&kgr;B could be prevented by a specific &bgr;2-antagonist. Our results demonstrate that dobutamine is a potent and specific inhibitor of NF-&kgr;B, and they thus provide a possible molecular mechanism for the immunomodulation associated with &bgr;-adrenergic therapy.