Dieter Kunz

Peripheral lipopolysaccharide administration transiently affects expression of brain-derived neurotrophic factor, corticotropin and proopiomelanocortin in mouse brain.

Peripheral inflammation induced by intraperitoneal (i.p.) injection of Lipopolysaccharide (LPS) is known to cause functional impairments in the brain affecting memory and learning. One of mechanisms may be the interference with neurotrophin (NT) expression and function. In the current study we administered a single, high dose of LPS (3mg/kg, i.p.) into mice and investigated changes in brain-derived neurotrophic factor (BDNF) gene expression within 1-6 days after LPS injection. Crude synaptosomes were isolated from brain tissue and subjected to Western-blot analyses. We found transient reductions in synaptosomal proBDNF- and BDNF protein expression, with a maximal decrease at day 3 as compared to saline injected controls. The time course of reduction of BDNF mRNA in whole brain extracts parallels the decrease in protein levels in synaptosomes. LPS effects in the central nervous system (CNS) are known to crucially involve the activation of the hypothalamic-pituitary-adrenal (HPA) axis. We analysed the time course of corticotropin releasing hormone (CRH)- and proopiomelanocortin (POMC) mRNA expression. As observed for BDNF-, CRH- and POMC mRNA levels are also significantly reduced on day 3 indicating a comparable time course. These results suggest that peripheral inflammation causes a reduction of trophic supply in the brain, including BDNF at synaptic sites. The mechanisms involved could be a negative feedback of the activated HPA axis.

Expression of nitric oxide synthase in rat glomerular mesangial cells mediated by cyclic AMP.

1 Treatment of rat mesangial cells with interleukin 1β (IL‐1β) or tumour necrosis factor α (TNFα) has been shown to induce a macrophage‐type of nitric oxide (NO) synthase. Here we report that adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) is another mediator that triggers induction of NO synthase in mesangial cells. 2 Incubation of mesangial cells with the β‐adrenoceptor agonist, salbutamol, forskolin or cholera toxin, which all activate adenylate cyclase and increase intracellular cyclic AMP concentration, increased nitrite formation in a dose‐dependent manner. Likewise, the addition of the membrane‐permeable cyclic AMP analogue, N6, 0–2′‐dibutyryladenosine 3′,5′‐phosphate (Bt2cyclic AMP) or the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine enhanced NO synthase activity in a dose‐dependent manner. 3 There was a lag period of about 8 h before a significantly enhanced secretion of nitrite could be detected upon exposure of cells to forskolin and for maximal stimulation, forskolin had to be present during the whole incubation period. 4 Treatment of mesangial cells with actinomycin D, cycloheximide or dexamethasone completely suppressed forskolin‐stimulated NO‐synthase activity, thus demonstrating that transcription and protein synthesis are necessary for nitrite formation. 5 Bt2 cyclic AMP, the most potent inducer of nitrite production, increased NO synthase mRNA levels in mesangial cells in a time− and dose‐dependent fashion. Dexamethasone completely inhibited the increase of NO synthase mRNA in response to Bt2 cyclic AMP. 6 Combination of Bt2 cyclic AMP and IL‐1β or TNFα revealed a strong synergy in terms of nitrite formation. Time‐course studies indicated that cyclic AMP needed to be increased during the whole period of IL‐1β stimulation for maximal nitrite production. 7 These observations suggest that cyclic AMP controls NO synthase expression in mesangial cells. Furthermore, the signalling cascades triggered by IL‐1β and TNFα synergize with the cyclic AMP pathway to stimulate NO synthase activity.

Cytokines and neurotrophins interact in normal and diseased states.

Abstract: Neurotrophins (NTs) such as nerve growth factor (NGF) as well as cytokines, for example, interleukin‐6 (IL‐6), are communicators between the nervous and immune systems. There is evidence for mutual interactions between NTs and cytokines. Strategies are being developed to elucidate the molecular mechanism/s of interactions and to understand how cytokines are involved in health and disease. Analysis of underlying signaling pathways in glial cells indicates that different transcription factors, such as NF‐κB, cAMP‐responsive‐element binding protein (CREB), and activator protein 1 (AP‐1), are involved in NT induction. IL‐6 and NTs of the NGF family are coexpressed at sites of nerve injury. Interactions of these factors could modulate both neuronal de‐ and regeneration: IL‐6 in conjunction with its soluble IL‐6 receptor induces a specific pattern of NTs in astrocytes in defined brain regions. This indicates that the IL‐6 system mediates a local supply of NTs that participate in diverse CNS functions, such as protection of neurons from insults, neuronal survival, and neuroimmune responses.

Expression of interleukin-6 and its receptor in the sciatic nerve and cultured Schwann cells: relation to 18-kD fibroblast growth factor-2

Expression of interleukin-6 (IL-6) and fibroblast growth factor-2 (FGF-2) in Schwann cells is modulated by external stimuli. To study possible interactions of both factors we have analyzed mutual effects of exogenous IL-6 and FGF-2 on the expression of each other and the corresponding receptor (R) molecules IL-6R and FGFR1 after peripheral nerve lesion in vivo and in vitro using cultured Schwann cells. Using rat Schwann cells we found that IL-6 did not exert any effects on the expression of FGF-2 and FGF receptor type 1 (R1) whereas exogenously applied 18-kD FGF-2 strongly increased the expression of the mRNAs of IL-6 and its receptor. In addition, immortalized Schwann cells over-expressing the 18-kD FGF-2 isoform showed elevated levels of IL-6 and IL-6R whereas immortalized Schwann cells over-expressing the high-molecular-weight isoforms (21 kD and 23 kD) displayed unaltered IL-6 and IL-6R expression levels. According to in situ hybridization studies of intact and crushed sciatic nerves in vivo, Schwann cells seems to be the main source of IL-6 and IL-6R. Following sciatic nerve crush, the FGF-2 and the IL-6 system are upregulated after the first hours. Furthermore, we showed that the early increase of the FGF-2 protein is mainly confined to the 18-kD isoform. These results are consistent with the idea of a functional coupling of FGF-2 and the IL-6 system in the early reaction of Schwann cells to nerve injury.

THERAPEUTIC STRATEGIES FOR THE INHIBITION OF INDUCIBLE NITRIC OXIDE SYNTHASE—POTENTIAL FOR A NOVEL CLASS OF ANTI-INFLAMMATORY AGENTS

In recent years, NO, a gas previously considered a potentially toxic chemical, has become established as a diffusible universal messenger mediating cell—cell communication throughout the body. In mammals, NO is a recognized mediator of blood vessel relaxation that helps to maintain blood pressure. In the central nervous system NO acts as a non‐conventional neurotransmitter and participates in the establishment of long‐term plasticity required for memory formation. In addition, NO is responsible for some parts of the host response to sepsis and inflammation and contributes to certain disease states. A number of strategies have emerged with regard to a pharmacological control of pathological NO overproductions. This review will discuss these novel therapeutic approaches that may provide new means for clinical medicine.

Cyclosporin derivatives inhibit interleukin 1β induction of nitric oxide synthase in renal mesangial cells

Treatment of mesangial cells with recombinant human interleukin 1 beta dose dependently increased nitrite formation due to the induction of a macrophage-type of nitric oxide (NO) synthase. Addition of cyclosporin A, cyclosporin G or cyclosporin H dose dependently inhibited interleukin 1 beta-induced nitrite generation. Half-maximal inhibition was observed at concentrations of 0.9 microM, 2.0 microM and 3.8 microM of cyclosporin A, cyclosporin G and cyclosporin H, respectively. Time-course studies indicated that cyclosporin A could be added up to 6 h after the interleukin 1 beta stimulus and still caused maximal inhibition of nitrite production. Furthermore, interleukin 1 beta increased NO synthase mRNA levels in mesangial cells and this effect was potently suppressed by all three cyclosporin derivatives. As cyclosporin H has no immunosuppressive activity, these data indicate that the inhibitory effect of the cyclosporin derivatives on NO synthase expression is not related to the immunosuppressive action of the drugs. This suggestion is further substantiated by the observation that the potent immunosuppressants rapamycin and FK506 did not alter interleukin 1 beta-induced NO synthase mRNA levels or nitrite generation in mesangial cells. In summary, these data demonstrate that cyclosporin derivatives potently modulate the L-arginine-NO pathway in renal mesangial cells.

Proteolytic cleavage of inducible nitric oxide synthase (iNOS) by calpain I

Proteolytic degradation of inducible nitric oxide synthase (iNOS or NOS2; EC 1.14.13.39) is one of the key steps by which the synthetic glucocorticoid dexamethasone controls the amount of iNOS protein and thus the production of nitric oxide (NO) in interferon-gamma-stimulated RAW 264.7 cells. In the present study we examined the role of the calmodulin (CaM)-binding site present within iNOS protein for the proteolytic degradation by the calcium-dependent neutral cysteine protease calpain I (EC 3.4.22.17). Using pulse chase experiments as well as cell-free degradation assays we show that the iNOS monomer is a direct substrate for cleavage by calpain I. Two structural determinants are involved in proteolytic cleavage, the canonical CaM-binding domain present at amino acids 501-532 and a conformational determinant located within iNOS. The access of the CaM-binding region appears to be critical for substrate cleavage as incubation of in vitro synthesized iNOS with purified CaM inhibits iNOS degradation by calpain I. Moreover, cytosolic CaM levels are decreased upon treatment of RAW 264.7 cells with dexamethasone as assessed by immunoprecipitation. The data shown herein provide novel insights into the underlying mechanisms involved in the anti-inflammatory actions of glucocorticoids.

Pyrrolidine dithiocarbamate differentially affects cytokine- and cAMP-induced expression of group II phospholipase A2 in rat renal mesangial cells

Renal mesangial cells express group II phospholipase A2 in response to two principal classes activating signals that may interact in a synergistic fashion. These two groups of activators comprise inflammatory cytokines such as interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNFα) and agents that elevate cellular levels of cAMP such as forskolin, an activator of adenylate cyclase. Using pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of nuclear factor NFκB, we determined its role in cytokine — and cAMP — triggered group II PLA2 expression. Micromolar amounts of PDTC suppress the IL‐1β‐ and TNF α‐dependent, but not the forskolin‐stimulated group II PLA2 activity in mesangial cells. Furthermore, PDTC inhibited the increase of group II PLA2 mRNA steady state levels in response to IL‐1β and TNFα, while only marginally affecting forskolin‐induced PLA2 mRNA level. Our data suggest that NFκB activation is an essential component of the cytokine signalling pathway responsible for group II PLA2 gene regulation and that cAMP triggers a separate signalling cascade not involving NFκB. These observations may provide a basis to study the underlying mechanisms involved in the regulation of group II PLA2 gene expression.

Suppression by cyclosporin A of interleukin 1β-induced expression of group II phospholipase A2 in rat renal mesangial cells

We investigated whether cyclosporin A, a potent immunosuppressive drug, affects group II phospholipase A2 (PLA2; EC 3.1.1.4) induction in rat renal mesangial cells. Previously we showed that the expression of group II PLA2 in rat renal mesangial cells is triggered by exposure of the cells to inflammatory cytokines such as interleukin 1β (IL‐1β) or tumour necrosis factor α and agents that elevate cellular levels of cyclic AMP. Treatment of mesangial cells with IL‐1β for 24 h induced PLA2 activity secreted into cell culture supernatants by about 16 fold. Incubation of mesangial cells with cyclosporin A inhibited IL‐1β‐induced PLA2 section in a dose‐dependent fashion, with an IC50 value of 4.3 μM. Cyclosporin A did not directly inhibit enzymatic activity of PLA2. Immunoprecipitation of radioactively labelled PLA2 protein from mesangial cell supernatants revealed that the inhibition of PLA2 activity is due to a suppression of PLA2 protein levels. This effect was preceded by a reduction of PLA2 mRNA steady state levels, as demonstrated by Northern blot analyses of total cellular RNA isolated from stimulated mesangial cells. In order to evaluate whether cyclosporin A would affect the transcriptional activity of the PLA2 gene, we performed nuclear run on transcription experiments and provided evidence that the transcription rate of the PLA2 gene is reduced by cyclosporin A. Previously we found that the nuclear transcription factor κB (NFκB) is an essential component of the IL‐1β‐dependent upregulation of PLA2 gene transcription. By electrophoretic mobility shift analysis, we demonstrated that cyclosporin A diminishes the formation of NFκB DNA‐binding complexes, thus suggesting that this transcription factor is a target for cyclosporin A‐mediated repression of PLA2 gene transcription. The data presented in this study strongly suggest that the cellular mechanism involved in the IL1β ‐ dependent transcriptional upregulation of the PLA2 gene in mesangial cells is a target for the action of cyclosporin A.

Expression profiling and Ingenuity biological function analyses of interleukin-6- versus nerve growth factor-stimulated PC12 cells

BackgroundThe major goal of the study was to compare the genetic programs utilized by the neuropoietic cytokine Interleukin-6 (IL-6) and the neurotrophin (NT) Nerve Growth Factor (NGF) for neuronal differentiation.ResultsThe designer cytokine Hyper-IL-6 in which IL-6 is covalently linked to its soluble receptor s-IL-6R as well as NGF were used to stimulate PC12 cells for 24 hours. Changes in gene expression levels were monitored using Affymetrix GeneChip technology. We found different expression for 130 genes in IL-6- and 102 genes in NGF-treated PC12 cells as compared to unstimulated controls. The gene set shared by both stimuli comprises only 16 genes.A key step is upregulation of growth factors and functionally related external molecules known to play important roles in neuronal differentiation. In particular, IL-6 enhances gene expression of regenerating islet-derived 3 alpha (REG3A; 1084-fold), regenerating islet-derived 3 beta (REG3B/PAPI; 672-fold), growth differentiation factor 15 (GDF15; 80-fold), platelet-derived growth factor alpha (PDGFA; 69-fold), growth hormone releasing hormone (GHRH; 30-fold), adenylate cyclase activating polypeptide (PACAP; 20-fold) and hepatocyte growth factor (HGF; 5-fold). NGF recruits GDF15 (131-fold), transforming growth factor beta 1 (TGFB1; 101-fold) and brain-derived neurotrophic factor (BDNF; 89-fold). Both stimuli activate growth-associated protein 43 (GAP-43) indicating that PC12 cells undergo substantial neuronal differentiation.Moreover, IL-6 activates the transcription factors retinoic acid receptor alpha (RARA; 20-fold) and early growth response 1 (Egr1/Zif268; 3-fold) known to play key roles in neuronal differentiation.Ingenuity biological function analysis revealed that completely different repertoires of molecules are recruited to exert the same biological functions in neuronal differentiation. Major sub-categories include cellular growth and differentiation, cell migration, chemotaxis, cell adhesion, small molecule biochemistry aiming at changing intracellular concentrations of second messengers such as Ca2+ and cAMP as well as expression of enzymes involved in posttranslational modification of proteins.ConclusionThe current data provide novel candidate genes involved in neuronal differentiation, notably for the neuropoietic cytokine IL-6. Our findings may also have impact on the clinical treatment of peripheral nerve injury. Local application of a designer cytokine such as H-IL-6 with drastically enhanced bioactivity in combination with NTs may generate a potent reparative microenvironment.