Signals regulating neurotrophin expression in glial cells.
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Animals↗
Biomedical subjects
P März
Publications and source records attributed to P März.
Human herpes virus 8 interleukin-6 homologue triggers gp130 on neuronal and hematopoietic cells.
Human herpes virus-8 (HHV8) encodes a cytokine named viral interleukin-6 (vIL-6) that shares 25% amino-acid identity with its human homologue. Human IL-6 is known to be a growth and differentiation factor of lymphatic cells and plays a potential role in the pathophysiology of various lymphoproliferative diseases. vIL-6 is expressed in HHV8-associated-diseases including Kaposi's sarcoma, Body-cavity-based-lymphoma and Castleman's disease, suggesting a pathogenetic involvement in the malignant growth of B-cell associated diseases and other malignant tumours. We expressed vIL-6 in Escherichia coli as a fusion protein with recombinant periplasmic maltose binding protein. After cleavage from the maltose binding protein moiety and purification, vIL-6 was shown to be correctly folded using circular dichroism spectroscopy. A rabbit antiserum was raised against the recombinant vIL-6 protein. vIL-6 turned out to be active on cells that expressed gp130 but no IL-6 receptor (IL-6-R) suggesting that, in contrast to human IL-6, vIL-6 stimulated gp130 directly. Accordingly, vIL-6 activity could be inhibited by a soluble gp130 Fc Fusion protein. vIL-6 was shown to induce neuronal differentiation of rat pheochromocytoma cells and to stimulate colony formation of human hematopoietic progenitor cells. Thus, vIL-6 exhibits biologic activity that has only been observed for the IL-6/soluble IL-6-R complex but not for IL-6 alone. These properties are important for the evaluation of the pathophysiological potential of vIL-6.
Amino Acid Sequence↗
Cytokines and neurotrophins interact in normal and diseased states.
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-kappa B, 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.
Animals↗
Interleukin-6 (IL-6) and its soluble receptor support survival of sensory neurons.
The cytokine interleukin-6 (IL-6) has multiple functions in the immune and hematopoietic systems. IL-6 is related to ciliary neurotrophic factor (CNTF), a trophic factor for motoneurons, sensory dorsal root ganglion (DRG) neurons, and other neuronal subpopulations. Both act via related receptor complexes, consisting of one ligand-specific alpha-receptor subunit (IL-6R and CNTFR, respectively) and two signal-transducing receptor components. Even though IL-6 is expressed by neurons and glia, the functions of IL-6 in the nervous system are poorly understood. Here, we report that exogenous human IL-6 promotes the survival of dissociated newborn rat DRG neurons in vitro if supplemented with soluble human IL-6-alpha-receptor. The dosages of human IL-6 and soluble human IL-6R necessary to achieve neurotrophic effects could be reduced markedly by linking ligand and alpha-receptor component in a designer cytokine. Furthermore, we show that newborn rat DRG neurons express and secrete bioactive IL-6. Endogenously secreted IL-6 does not enhance survival of these neurons in vitro, suggesting that DRG neurons do not sufficiently express cell surface IL-6R. Exogenously added soluble rat IL-6R rendered DRG neurons responsive to secreted IL-6. Our results indicate an autocrine function of IL-6 in DRG neuron survival which depends on membrane-bound or soluble IL-6R as a neurotrophic cofactor.
Animals↗
Role of interleukin-6 and soluble IL-6 receptor in region-specific induction of astrocytic differentiation and neurotrophin expression.
Increasing evidence supports an essential role for interleukin-6 (IL-6) in the development, differentiation, as well as de- and re-generation of neurons in the central nervous system (CNS). Both IL-6 and its specific receptor (IL-6R) are expressed on neurons and glial cells including astrocytes. In this study, we have analyzed the responses of primary rat astrocytes of various brain regions to IL-6 with respect to morphological changes and neurotrophin expression. Since IL-6 alone failed to initiate effects on astrocytes, we have examined whether the soluble IL-6R (sIL-6R) can modulate the responsiveness of to IL-6 in these cells. For this purpose, we used a highly active fusion protein of IL-6 and sIL-6R, which is designated Hyper-IL-6 (H-IL-6). We show that treatment of cultured astrocytes with Hyper-IL-6 promotes region-specific morphological changes of GFAP-positive astrocytes from typical stellate- to fibrous-like cells. In addition, we find that Hyper-IL-6 induces expression of neurotrophins (NTs) of the nerve growth factor (NGF)-family in a dose-dependent manner. Interestingly, astrocytes of various brain regions show differing patterns of cytokine-induced NT expression: NGF is maximally induced in cortex and hippocampus, NT-3 in hippocampus, and NT-4/5 in cortex and cerebellum. In summary, our results indicate that IL-6 in conjunction with sIL-6R regulates specific neurotrophin expression in astrocytes in a brain region dependent manner. Thus, the IL-6 system provides a local supply of neurotrophins that participate in diverse CNS functions such as protection of neurons from insults, neuronal survival, and neuro-immune responses.
Animals↗
Generation and function of the soluble interleukin-6 receptor.
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Alternative Splicing↗
Neural activities of IL-6-type cytokines often depend on soluble cytokine receptors.
Cytokines of the interleukin-6 (IL-6) family participate in regulatory and inflammatory processes within the nervous system. IL-6, ciliary neurotrophic factor (CNTF) and IL-11 act via specific membrane receptors which, together with their ligands, associate with signal-transducing receptor subunits thereby initiating cytoplasmic signalling. Cells which only express signal-transducing receptor subunits but no ligand binding subunits for IL-6, CNTF and IL-11 are refractory to these cytokines. An unusual feature of the IL-6 cytokine family is that the soluble forms of the ligand binding receptor subunits generated by one cell type in complex with their ligands can directly stimulate the signal-transducing receptor subunits on different cell types which lack ligand binding receptor subunits. This process has been named transsignalling. This article focuses on the importance of transsignalling events in neuronal differentiation and survival responses.
Animals↗
The IL-6/sIL-6R fusion protein hyper-IL-6 promotes neurite outgrowth and neuron survival in cultured enteric neurons.
The undisturbed development of the enteric nervous system depends on the supply of various neurotrophic factors during ontogenesis. Besides glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) take part in its development. CNTF and LIF belong to the interleukin-6 (IL-6) family of cytokines. The combination of IL-6 and the soluble IL-6 receptor accelerates peripheral nerve regeneration. In this study, we examined the effect of the fusion protein Hyper-IL-6, which consists of IL-6 and the soluble receptor sIL-6R, on neurite outgrowth and neuronal survival in vitro. Myenteric plexus of newborn rats was dissected and dissociated. Cells were grown in either serum-free chemically defined medium alone or medium supplemented with sIL-6R, IL-6, sIL-6+IL-6, Hyper-IL-6, CNTF, LIF, or GDNF. Average neurite outgrowth per neuron was highest in GDNF-treated and Hyper-IL-6-treated cultures. The number of neurite-bearing neurons was reduced in GDNF cultures compared with Hyper-IL-6-treated cells, so that the total neurite outgrowth was maximal after Hyper-IL-6 stimulation. Hyper-IL-6 furthermore stimulated neuronal survival and morphologic differentiation of the enteric glia.
Animals↗
Sympathetic neurons can produce and respond to interleukin 6.
Neuronal expression of cytokines is an area of active investigation in the contexts of development, disease, and normal neural function. Although cultured rat sympathetic neurons respond very weakly to exogenous interleukin 6 (IL-6), we find that addition of soluble IL-6 receptor (sIL-6R) and IL-6 enhances neuronal survival in the absence of nerve growth factor. Neutralizing monoclonal antibodies against IL-6 block these effects. Addition of IL-6 and sIL-6R also induces a subset of neuropeptide and transmitter synthetic enzyme mRNAs identical to that demonstrated for leukemia inhibitory factor, ciliary neurotrophic factor, and oncostatin M. Both of these effects are duplicated by addition of a highly active fusion protein of sIL-6R and IL-6, covalently linked by a flexible peptide chain, which is designated H-IL-6. In addition, we show that sympathetic neurons produce IL-6. In situ hybridization indicates a neuronal localization of IL-6 mRNA in superior cervical ganglia, and bioactive IL-6 protein is detected in ganglion culture supernatants. Interestingly, the IL-6 produced by sympathetic neurons does not lead to survival of these cells in culture unless sIL-6R is added. Thus, sympathetic neurons can produce IL-6 and may respond to it in an autocrine/paracrine manner if sIL-6R is present. Moreover, the prior findings of sIL-6R in serum and inflammatory fluids now have added interest in the context of neuro-immune interactions.
3T3 Cells↗
Interleukin-6 (IL-6) and soluble forms of IL-6 receptors are not altered in cerebrospinal fluid of Alzheimer's disease patients.
We quantitated interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R) and soluble form of the IL-6 signal-transducing protein gp130 (sgp130) in cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD) (n = 17) and control subjects (n = 18) using sensitive enzyme-linked immunosorbent assays (ELISA). Our results show that none of the parameters examined was significantly different in CSF of AD patients as compared to control age-matched non-demented patients. We conclude that CSF levels of IL-6 and their soluble receptors do not necessarily reflect local changes of the IL-6 system that has been shown to be involved in neurodegenerative events occurring in AD. Levels of sgp130 are substantially high (approximately 100 ng/ml) in the CSF of all individuals probably representing a high antagonistic potential.
Aged↗
Activation of gp130 by IL-6/soluble IL-6 receptor induces neuronal differentiation.
Interleukin-6 (IL-6) on target cells binds to the specific IL-6 receptor (IL-6R) and subsequently induces homodimerization of the signal-transducing protein gp130. Cells which express gp130 but no IL-6R and which therefore do not respond to IL-6 can be stimulated by the complex of IL-6 and soluble IL-6R (slL-6R). Here we show that on rat pheochromocytoma cells (PC12), the combination of IL-6 and slL-6R but not IL-6 alone induces expression of c-fos, GAP-43 and neuron-specific enolase followed by neuron-specific differentiation and formation of a neuronal network. The differentiation was dose-and time-dependent and followed the same kinetics as nerve-growth factor (NGF)-induced differentiation. The responses of PC12 cells to IL-6/sIL-6R and NGF were additive, suggesting independent signaling pathways. We demonstrate that activation of gp130 generates a neuronal differentiation signal that is equivalent to and independent of trk/NGF receptor tyrosine kinase. Interestingly, the failure of IL-6 to induce differentiation of PC12 cells is not due to lack of surface expression of IL-6R as IL-6 alone triggered expression of GAP-43 mRNA and protein. We hypothesize that PC12 cells express more gp130 than IL-6R and that the extent of activated gp130 molecules determines the quality of the response.
Animals↗
Alternative assay procedures for cytokines and soluble receptors of the IL-6 family.
Human hepatoma cells (HepG2 cells) were transfected with expression vectors for human IL-6 (hIL-6) and rat IL-6R (rIL-6-R). The cell lines were used for testing the biological activity of different IL-6 species, soluble hIL-6R (shIL-6R) and some members of the IL-6 cytokine family by means of an ELISA procedure. The assay is based on induction of the gene expression of the acute phase protein haptoglobin in hepatoma cells and provides an alternative bioassay taking advantage of the hepatocyte stimulatory activity of IL-6 (as opposed to the B9 proliferative assay). A dose-response experiment with IL-6 showed that half-maximal stimulation was achieved with approx. 5 ng/ml of hIL-6 in HepG2 cells and with 5-10 ng/ml muIL-6 in HepG2-rIL-6R cells after 24 h. The same response was achieved with 10 ng/ml shIL-6R in HepG2-IL6 cells. In conclusion, the assay is fast and reliable and might be adopted for other cytokines and receptors with hepatocyte stimulating activity.
Animals↗
Expression of interleukin-6 receptor (IL-6R) and gp130 mRNA in PC12 cells and sympathetic neurons: modulation by tumor necrosis factor alpha (TNF-alpha).
Recent findings indicate that IL-6, besides its various biological effects, also exerts neurotrophic and neuroprotective functions. Using the pheochromocytoma cell line PC12 and cultured primary sympathetic neurons, we investigated whether neurons express the IL-6 receptors, IL-6R and gp130, and how they might be regulated. For these studies we used RT-PCR and in situ hybridization. We provide here evidence for the expression of functional IL-6Rs in peripheral sympathetic neurons and PC12 cells. Furthermore we demonstrate that cytokines modulate the expression of IL-6R and gp130 mRNA. This modulation is much more pronounced in neuronally-differentiated PC12 cells than in undifferentiated cells. Among various cytokines tested, tumor necrosis factor alpha (TNF-alpha) turned out to be a major regulator of the IL-6R and gp130 mRNA expression. The induction was time- and dose-dependent for both genes. Maximal induction was reached within 16 h at a concentration of 0.1 nM TNF-alpha. The stimulatory effect of TNF-alpha on the IL-6R system was completely inhibited by the simultaneous addition of the glucocorticoid dexamethasone. In summary, our results show that sympathetic neurons and neuron-like differentiated PC12 cells express functional IL-6R and gp130, and that the expression of their mRNAs is modulated by cytokines. We suggest that cytokines such as IL-6 can modulate sympathetic neuron function.
Animals↗
[Reply to the comments by H. Hutten. "Comparative evaluation of equipment: the problem of false positives"].
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Anesthesiology↗
[In electric nerve stimulation can the distance from the nerves be inferred from the intensity of muscle contraction? Possible parameters and sources of error].
Electrical nerve stimulation is a useful tool in regional anesthesia; it assists in locating the nerves. This investigation deals with electrical resistances at adhesive electrodes attached to the skin. The influence of external electrical resistance on the stimulating impulse is demonstrated. A new type of nerve stimulator is described; which integrates a measuring device that indicates the electrical impulse actually flowing in the patient. With this device, relationships between stimulating impulse, intensity of muscle contraction, and distance of the puncture cannula from the nerve can be assessed. MATERIALS AND METHODS. The electrical curve I = f (R) was determined using 3 different nerve stimulators (R = 1 - 15k omega). On each of five test persons, five adhesive electrodes were attached to the skin 10 cm apart. The resistance was assessed between these electrodes and a subcutaneously inserted puncture needle. The right and left brachial plexuses of the 5 subjects were punctured, using the axillary approach. A synchronized video camera simultaneously recorded the following values: (1) electrical impulse; (2) corresponding muscle contraction; and (3) the position of the puncture cannula. A scale ranging from 0-5 was applied to define the strength of the muscle contractions. RESULTS. The external resistance as measured under clinical conditions may limit the output impulse of nerve stimulators. In this case, the exerting impulse is lower than that indicated by the appliance. This error can only be identified using an impulse-measuring device. Nerve stimulators not equipped with a measuring device increase the risk of malpuncture, potentially resulting in nerve lesions. Skin resistance at adhesive electrodes varied from patient to patient within a range of 1.1 to 8.2 K omega. Preparing the skin appropriately (wiping with sandpaper) decreased the resistance by only 6% during the first 30 min. Within a distance of 50 cm to the puncture needle, the position of the adhesive electrode did not play a significant role. Axillary puncture of the brachial plexus resulted in the following values: (1) A stimulating impulse (cross-wave) of 1 mA and 1 ms exerted at a distance of 4 mm to the nerve induced a contraction of strength 3. (2) Reducing the impulse at this site by one-half (0.55 mA) resulted in just-visible contractions (strength 1). (3) Advancing the cannula at a stimulating impulse of 0.55 mA inside the neurovascular sheath again produced contractions of strength 3. (4) Performing the puncture with a blunt needle and tracing a distinct resistance, the impulse of 0.6 mA elicited contractions of strength 2. The needle tip was still outside the vascular nerve sheath. If this resistance was overcome and the needle tip lay inside the neurovascular sheath, the impulse could be reduced by one-half (0.32 mA) to produce contractions of strength 2.
Anesthesia, Conduction↗
[A new catheter set for continuous axillary plexus anesthesia].
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Anesthesia, Local↗
[Electrical nerve localization and catheter technic. A safe method for brachial plexus anesthesia].
A technique for sub-axillary blockade of the brachial plexus by means of a special catheter set is presented. A plastic self-retaining cannula is placed within the vasomotor nerve sheath, using the advantages of electric nerve stimulation. In our opinion the decisive advantages of this procedure lie in the fact that the user is not dependent on the cooperation of the patient and anatomical orientation takes place on the basis of clear and objective criteria through the muscle contractions induced in this way. If required, a catheter can be inserted into the vasomotor nerve sheath, without any additional expenditure of time, through the small-lumen puncture cannula by the Seldinger technique. If this procedure is carried out properly there is practically no danger of neural and vascular injuries. This method makes subsequent injections into the vasomotor nerve sheath through repeated puncture unnecessary. The catheter technique makes it possible to operate on the upper extremity using regional anaesthesia, whose effects can be prolonged beyond those of long-acting anaesthetics. The possibility of incomplete blockade resulting from individual variations in the amounts of local anaesthetics needed, is excluded and the anaesthetist can, if necessary, carry out pre- and post-operative pain therapy.
Anesthesia, Conduction↗
[Continuous blockade of the lumbar plexus ("3-in-1 block") in perioperative pain therapy].
This is a description of a technique for a continuous blockade of the lumbar plexus by means of a specially developed catheter set. The femoral nerve is located more or less atraumatically, on the basis of objective criteria, by the use of a nerve stimulator and a small-lumen puncture cannula. The catheter is subsequently inserted into the fascia surrounding the nerves by the Seldinger technique. The advantage of this catheter technique lies in the fact that the normal blockade period can be extended at will, without any great increase in the amount of time and material needed. Pain therapy for the patient during the operation does not involve the necessity of further punctures with the risk of neural lesion resulting from the fact that partial anaesthesia still exists. Individual dosage is possible because of the anatomical circumstances. The catheter technique we used enabled us in many cases to reduce the amount of local anaesthetic used. No disturbances in the functioning of the bladder or vasomotor system, as is the case with spinal conduction anaesthetics, were observed. In the pre-operative phase this 3-in-1 blockade procedure facilitates the treatment of patients with a fractured collum femoris in particular, and after an operation it makes it possible to mobilize the patient sooner, without the administration of additional analgetics.
Anesthesia, Spinal↗