Alexandra K. Kiemer

Inhibition of p38 MAPK activation via induction of MKP-1 : atrial natriuretic peptide reduces TNF-alpha-induced actin polymerization and endothelial permeability

The atrial natriuretic peptide (ANP) is a cardiovascular hormone possessing antiinflammatory potential due to its inhibitory action on the production of inflammatory mediators, such as tumor necrosis factor-&agr; (TNF-&agr;). The aim of this study was to determine whether ANP is able to attenuate inflammatory effects of TNF-&agr; on target cells. Human umbilical vein endothelial cells (HUVECs) were treated with TNF-&agr; in the presence or absence of ANP. Changes in permeability, cytoskeletal alterations, phosphorylation of p38 MAPK and HSP27, and expression of MKP-1 were determined by macromolecule permeability assay, fluorescence labeling, RT-PCR, and immunoblotting. Antisense studies were done by transfecting cells with MKP-1 antisense oligonucleotides. Activation of HUVECs with TNF-&agr; lead to a significant increase of macromolecule permeability and formation of stress fibers. Treatment of cells with ANP (10−8 to 10−6 mol/L) significantly reduced the formation of stress fibers and elevated permeability. Both TNF-&agr;–induced effects were shown to be mediated via the activation of p38 using SB203580, a specific inhibitor of p38. ANP significantly reduced the TNF-&agr;–induced activation of p38 and attenuated the phosphorylation of HSP27, a central target downstream of p38. ANP showed no effect on p38 upstream kinases MKK3/6. However, a significant induction of the MAPK phosphatase MKP-1 mRNA and protein could be observed in ANP-treated cells. Antisense experiments proved a causal role for MKP-1 induction in the ANP-mediated inhibition of p38. These data show the inhibitory action of ANP on TNF-&agr;–induced changes in endothelial cytoskeleton and macromolecule permeability involving an MKP-1–induced inactivation of p38 MAPK. These effects point to an antiinflammatory and antiatherogenic potential of this cardiovascular hormone.

Silibinin protects mice from T cell-dependent liver injury.

BACKGROUND/AIMS Silibinin is the major pharmacologically active compound of the Silybum marianum fruit extract silymarin. Its well-known hepatoprotective activities are mostly explained by antioxidative properties, inhibition of phosphatidylcholine synthesis or stimulation of hepatic RNA and protein synthesis. Here, we characterized the hepatoprotective potential of silibinin as an immune-response modifier in T cell-dependent hepatitis in vivo. METHODS Silibinin was tested in the mouse model of concanavalin A (ConA)-induced, T cell-dependent hepatitis. Liver injury was assessed by quantification of plasma transaminase activities and intrahepatic DNA fragmentation. Plasma cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA), intrahepatic cytokine and inducible NO synthase (iNOS) mRNA levels by reverse transcriptase polymerase chain reaction, intrahepatic iNOS expression by immunofluorescent staining, and intrahepatic nuclear factor kappa B (NF-kappaB) activation by electrophoretic mobility shift assay. RESULTS Silibinin significantly inhibited ConA-induced liver disease. Silibinin proved to be an immune-response modifier in vivo, inhibiting intrahepatic expression of tumor necrosis factor, interferon-gamma, interleukin (IL)-4, IL-2, and iNOS, and augmenting synthesis of IL-10. In addition, silibinin inhibited intrahepatic activation of NF-kappaB. CONCLUSIONS Silibinin, suppressing T cell-dependent liver injury as an immune-response modifier, might be a valuable drug in therapeutic situations in which intrahepatic immunosuppression is required.

cGMP-Mediated Inhibition of TNF-α Production by the Atrial Natriuretic Peptide in Murine Macrophages

The atrial natriuretic peptide (ANP) is suggested to regulate inflammatory response by alteration of macrophage functions. The aim of this study was to investigate whether ANP influences production of TNF-α. TNF-α production in murine bone marrow-derived macrophages was induced by LPS, and TNF-α secretion (±ANP) was determined by L929 bioassay. ANP dose dependently (10−8–10−6 M) inhibited TNF-α release by up to 95%. The effect was mediated via the guanylate cyclase-coupled A receptor, as was shown by employing dibutyryl-cGMP, the cGMP-inhibitory compound Ly-83583, and the A receptor antagonist HS-142-1. A specific ligand of the natriuretic peptide “clearance” receptor inhibited TNF-α production only at 10−7 and 10−8 M, but not at 10−6 M. The B receptor ligand C-type natriuretic peptide showed no TNF-α-inhibitory effect. To investigate the underlying mechanism of ANP-mediated TNF-α inhibition, Northern blot was performed. ANP-treated macrophages displayed decreased TNF-α-mRNA levels. Besides the known inhibition of NF-κB activation, in this study we demonstrated that ANP also attenuates the activation of the proinflammatory transcription factor AP-1 (gel shift assay). ANP did not alter subunit composition of AP-1 complexes, as was shown by supershift assays applying anti-c-jun and anti-c-fos Abs. To get information on the ANP effect for human inflammatory processes, we investigated cytokine production in human LPS-activated blood. ANP significantly attenuated production of TNF-α and IL-1β without affecting production of IL-10 and IL-1ra. In summary, ANP was shown to attenuate TNF-α production of LPS-activated macrophages via cGMP. The inhibition is suggested to involve transcriptional processes that are the result of reduced activation of responsible transcription factors.

Phyllanthus amarus has anti-inflammatory potential by inhibition of iNOS, COX-2, and cytokines via the NF-κB pathway

BACKGROUND/AIMS Phyllanthus amarus is a herbal medicine traditionally applied in the treatment of viral hepatitis. Aim of this study was to investigate potential anti-inflammatory properties of standardized P. amarus extracts concerning a potential influence of P. amarus on endotoxin-induced nitric oxide synthase (iNOS), cyclooxygenase (COX-2), and cytokine production in vivo and in vitro. METHODS Investigations were performed in rat Kupffer cells (KC), in RAW264.7 macrophages, in human whole blood, and in mice. Cells were stimulated with lipopolysaccharides (LPS) in the presence or absence of P. amarus extracts (hexane, EtOH/H(2)O), mice were treated with galactosamine/LPS as a model for acute toxic hepatitis. Nitrite was measured by Griess assay, prostaglandin E(2) (PGE(2)) by radioimmunoassay, and cytokines by enzyme-linked immunosorbent assay. iNOS and COX-2 were determined by Western blot, activation of NF-kappaB and AP-1 by EMSA. RESULTS P. amarus EtOH/H(2)O and hexane extracts showed an inhibition of LPS-induced production of NO and PGE(2) in KC and in RAW264.7. The extracts also attenuated the LPS-induced secretion of tumor necrosis factor (TNF-alpha) in RAW264.7 as well as in human whole blood. Both extracts reduced expression of iNOS and COX-2 and inhibited activation of NF-kappaB, but not of AP-1. P. amarus inhibited induction of interleukin (IL)-1beta, IL-10, and interferon-gamma in human whole blood and reduced TNF-alpha production in vivo. CONCLUSIONS This work shows that standardized extracts of P. amarus inhibit the induction of iNOS, COX-2, and TNF-alpha. Therefore, we report for the first time an anti-inflammatory potential of this traditionally employed herbal medicine both in vitro and in vivo.

Autocrine Regulation of Inducible Nitric-oxide Synthase in Macrophages by Atrial Natriuretic Peptide

Atrial natriuretic peptide (ANP), a cardiovascular hormone, has been shown to inhibit synthesis of nitric oxide in lipopolysaccharide (LPS)-activated mouse bone marrow-derived macrophages via activation of its guanylate cyclase-coupled receptor. The goal of the present study was to elucidate the potential sites of inducible nitric-oxide synthase (iNOS) regulation affected by ANP and revealed the following. 1) ANP and dibutyryl-cGMP did not inhibit catalytic iNOS activity measured by the conversion rate ofl-[3H]arginine tol-[3H]citrulline in homogenates of LPS-treated cells. 2) Pretreatment of cells with ANP dose-dependently reduced the LPS-inducedl-[3H]citrulline production that has been shown to be due to reduced iNOS protein levels detected by Western blot. 3) ANP does not alter the ratio of catalytically active iNOS dimer versus inactive iNOS monomer considered to be a major post-translational regulatory mechanism for the enzyme. 4) Macrophages exposed to ANP display decreased LPS-induced iNOS mRNA levels. 5) Importantly, two basic mechanisms seem to be responsible for this observation, i.e. ANP specifically induced acceleration of iNOS mRNA decay and ANP reduced binding activity of NF-κB, the transcription factor predominantly responsible for LPS-induced iNOS expression in murine macrophages. Moreover, 6) ANP acts via an autocrine mechanism since recently ANP was shown to be secreted by LPS-activated macrophages, and we demonstrated here that LPS-induced NO synthesis was increased after blocking the binding of endogenous ANP by a receptor antagonist. These observations suggest ANP as a new autocrine macrophage factor regulating NO synthesis both transcriptionally and post-transcriptionally. ANP may help to balance NO production of activated macrophages and thus may allow successful immune response without adverse effects on host cells.

Effect of allicin and ajoene, two compounds of garlic, on inducible nitric oxide synthase

Inducible nitric oxide synthase (iNOS) has recently been shown to be present in human atherosclerotic lesions and to promote the formation of deleterious peroxynitrite. Allicin and ajoene are discussed as active compounds with regard to the beneficial effects of garlic in atherosclerosis. The aim of this study was to investigate the effect of allicin and ajoene on the iNOS system in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Ajoene (IC50 2.5-5 microM) and allicin (IC50 15-20 microM) dose dependently reduced nitrite accumulation, a parameter for NO synthesis, in supernatants of LPS-stimulated (1 microg/ml, 20 h) macrophages. Accordingly, reduced iNOS enzyme activities were measured by conversion of L-[3H]arginine to L-[3H]citrulline in homogenates of LPS-activated cells treated with ajoene or allicin. None of these compounds, however, showed a direct effect on the catalytic-activity of iNOS. Consequently, iNOS protein and mRNA expression in ajoene (10 microM) or allicin (50 microM) treated cells were evaluated by Western blot and Northern blot analysis, respectively. Markedly reduced iNOS protein as well as mRNA levels were demonstrated. These observations indicate that allicin and ajoene inhibit the expression of iNOS in activated macrophages. The possible link of this effect to the beneficial features attributed to garlic is discussed.

Atrial Natriuretic Peptide Induces Mitogen-Activated Protein Kinase Phosphatase-1 in Human Endothelial Cells via Rac1 and NAD(P)H Oxidase/Nox2-Activation

The cardiovascular hormone atrial natriuretic peptide (ANP) exerts anti-inflammatory effects on tumor necrosis factor-&agr;–activated endothelial cells by inducing mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). The underlying mechanisms are as yet unknown. We aimed to elucidate the signaling pathways leading to an induction of MKP-1 by ANP in primary human endothelial cells. By using antioxidants, generation of reactive oxygen species (ROS) was shown to be crucially involved in MKP-1 upregulation. ANP was found to increase ROS formation in cultured cells as well as in the endothelium of intact rat lung vessels. We applied NAD(P)H oxidase (Nox) inhibitors (apocynin and gp91ds-tat) and revealed this enzyme complex to be crucial for superoxide generation and MKP-1 expression. Moreover, by performing Nox2/4 antisense experiments, we identified Nox2 as the critically involved Nox homologue. Pull-down assays and confocal microscopy showed that ANP activates the small Rho-GTPase Rac1. Transfection of a dominant-negative (RacN17) and constitutively active Rac1 mutant (RacV12) indicated that ANP-induced superoxide generation and MKP-1 expression are mediated via Rac1 activation. ANP-evoked production of superoxide was found to activate c-Jun N-terminal kinase (JNK). Using specific inhibitors, we linked ANP-induced JNK activation to MKP-1 expression and excluded an involvement of protein kinase C, extracellular signal-regulated kinase, and p38 MAPK. MKP-1 induction was shown to depend on activation of the transcription factor activator protein-1 (AP-1) by using electrophoretic mobility shift assay and AP-1 decoys. In summary, our work provides insights into the mechanisms by which ANP induces MKP-1 and shows that ANP is a novel endogenous activator of endothelial Rac1 and Nox/Nox2.

Inhibition of LPS-induced nitric oxide and TNF-α production by α-lipoic acid in rat Kupffer cells and in RAW 264.7 murine macrophages

The activation of Kupffer cells represents a central mechanism of inflammatory liver injury involving the production of two important inflammatory mediators, nitric oxide and TNF‐α. The aim of this study was to investigate the effect of the hepatoprotective compound α‐lipoic acid (thioctic acid) on the production of nitric oxide and TNF‐α in isolated rat Kupffer cells and RAW 264.7 macrophages. Isolated rat Kupffer cells or RAW 264.7 were either untreated, treated with α‐lipoic acid (500 µg/mL), or activated with 1 µg/mL of lipopolysaccharide in the presence or absence of α‐lipoic acid (0.2−500 µg/mL). After 20 h the accumulation of nitrite was measured by the Griess assay. Tumour necrosis factor‐α secretion was quantified after 4 h by L929 bioassay. Cell viability was determined by mitochondrial reduction of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) test, nuclear factor‐κB (NF‐κB) and activator protein‐1 (AP‐1) DNA binding activity by gelshift assays. Treatment of Kupffer cells and RAW 264.7 with α‐lipoic acid alone had no effect on basal nitric oxide production. However, α‐lipoic acid significantly inhibited lipopolysaccharide‐induced nitrite accumulation. α‐Lipoic acid did not alter basal TNF‐α secretion in Kupffer cells, whereas it significantly inhibited lipopolysaccharide‐induced TNF‐α production. α‐Lipoic acid attenuated the activation of nuclear factor‐κB and AP‐1, two transcription factors pivotal in induction of inducible nitric oxide synthase and TNF‐α. α‐Lipoic acid significantly inhibits lipopolysaccharide‐induced macrophage production of nitric oxide and TNF‐α via an attenuated activation of NF‐κB and activator protein‐1. The reduced production of nitric oxide and TNF‐α in Kupffer cells may be involved in the hepatoprotective action conveyed by α‐lipoic acid.

MAPK phosphatase-1 represents a novel anti-inflammatory target of glucocorticoids in the human endothelium

Glucocorticoids are well‐established anti‐inflammatory drugs thought to mainly act by inhibition of proinflammatory transcription factors like NFKB. In recent years, however, transcription factor‐independent mechanisms of glucocorticoid action have been proposed, namely the influence on MAPK pathways. Here we identify MAPK phosphatase‐1 (MKP‐1) as a pivotal mediator of the anti‐inflammatory action of glucocorticoids in the human endothelium. We applied dexamethasone (Dex) to TNF‐a‐activated human endothelial cells and used the adhesion molecule E‐selectin as inflammatory read‐out parameter. Dex is known to reduce the expression of E‐selectin, which is largely regulated by NF‐κB. Here, we communicate that Dex at low concentrations (1–100 nM) markedly attenuates E‐selectin expression without affecting NF‐κB. Importantly, Dex is able to increase the expression of MKP‐1, which causes an inactivation of TNF‐α‐induced p38 MAPK and mediates inhibition of E‐selectin expression. In endothelial MKP‐1‐/‐cells differentiated from MKP‐1‐/‐embryonic stem cells and in MKP‐1‐silenced human endothelial cells, Dex did not inhibit TNF‐α‐evoked E‐selectin expression. Thus, our findings introduce MKP‐1 as a novel and crucial mediator of the anti‐inflammatory action of glucocorticoids at low concentrations in the human endothelium and highlight MKP‐1 as an important and promising anti‐inflammatory drug target. Fürst, R., Schroeder, T., Eilken, H. M., Bubik, M. F., Kiemer, A. K., Stefan Zahler, S., Vollmar, A. M. MAPK phosphatase‐1 represents a novel anti‐inflammatory target of glucocorticoids in the human endothelium FASEB J. 21, 74–80 (2007)

Atrial natriuretic peptide reduces expression of TNF-α mRNA during reperfusion of the rat liver upon decreased activation of NF-κB and AP-1

Abstract Background/Aims: The cardiovascular hormone Atrial Natriuretic Peptide (ANP) attenuates activation of the pro-inflammatory transcription factor NF-κB in macrophages. ANP was also shown to protect from ischemia-reperfusion injury of the rat liver. This study aimed to investigate the effects of this immunomodulatory hormone and its second messenger cGMP on the activation of the two redox-sensitive transcription factors AP-1 and NF-κB and the expression of corresponding pro-inflammatory target genes during ischemia and reperfusion of the liver. The identification of the mechanisms underlying the protection by ANP should reveal new aspects concerning the pathomechanisms of ischemia/reperfusion injury. Methods: Rat livers were perfused with and without ANP or 8-Br-cGMP preceding 24 h of cold storage in University of Wisconsin solution. During reperfusion NF-κB and AP-1 DNA binding activities were determined in freeze-clamped liver samples by electrophoretic mobility shift assay. Protein levels of p50, p65, and of IκB were determined by Western blot. mRNA coding for inducible nitric oxide synthase, cyclooxygenase-2, and TNF-α was determined by RT-PCR and Northern blot. Results: After 45 min of reperfusion DNA binding activities of NF-κB were increased, whereas in ANP pre-treated livers this effect was markedly reduced. AP-1, another important redox-sensitive transcription factor, was activated and in the course of reperfusion the subunit composition of AP-1 changed as assessed by supershift assays. ANP markedly reduced binding activities of both forms of AP-1. 8-Br-cGMP mimicked the effects of ANP on NF-κB and AP-1. Neither inducible nitric oxide synthase nor cyclooxygenase-2 mRNA could be detected. In contrast, a profound expression of transcripts coding for TNF-α was detected in the course of reperfusion and ANP markedly reduced TNF-α mRNA expression. Conclusion: ANP seems to mediate its protective effect during ischemia and reperfusion by reducing the activation of NF-κB and AP-1 via cGMP. The reduced binding activity of these redox-sensitive transcription factors was accompanied by a diminished mRNA expression of TNF-α, a cytokine known to be involved in cellular damage in ischemia reperfusion injury.