Interleukin-1 beta and interferon-gamma induce proliferation and apoptosis in cultured Schwann cells

This study reports that in Schwann cell tissue culture the administration of the two pro-inflammatory cytokines, interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma), at different dosages, singly or in combination, can induce apoptosis and/or mitosis.Schwann cell apoptosis was maximal within 24 h of stimulation with 50 U/ml of IFN-gamma, while proliferation was at its peak within 24 h with 10 U/ml IL-1 beta, and both processes decreased progressively by 48 and 72 h. Moreover, the combination of the two cytokines did not show any synergistic effect. These data can be interpreted as a possible involvement of pro-inflammatory cytokines not only in myelin disruption but also in promoting remyelination.     

Linomide (roquinimex) affects the balance between pro- and anti-inflammatory cytokines in vitro in multiple sclerosis

Objectives - Multiple sclerosis (MS) is characterized by high levels of circulating mononuclear cells (MNC) that respond to myelin proteins like myelin basic protein (MBP) in vitro by expressing mRNA of both pro-inflammatory cytokines, e.g. interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and lymphotoxin (LT) that may make MS worse, and anti-inflammatory cytokines like IL-4, IL-10 and transforming growth factor-β (TGF-β) that may act beneficially. Substances that down-regulate cytokines such as TNF-α or promote IL-10 or TGF-β can be anticipated to affect MS beneficially. Material and methods - In situ hybridization to detect and enumerate IFN-γ, TNF-α, LT, IL-4, IL-10 and TGF-β mRNA expressing blood MNC after stimulation with myelin basic protein (MBP), control antigens and without antigen in presence and absence of Linomide (roquinimex, LS-2616) was employed. In parallel, ELISPOT assay to detect MBP- and PHA-reactive IFN-γ secreting blood MNCLinomide was used. Results - Here we report that Linomide, a synthetic immunomodulator, at concentrations effective in vivo reduces the number of MBP-reactive TNF-a and increases MBP-reactive IL-10 and TGF-β mRNA expressing MNC from MS patients'blood when analysed in vitro. Compared to dexamethasone, Linomide up-regulated levels of blood MNC expressing mRNA of TGF-β after culture in presence of MBP. Conclusions - Changes of cytokine balance towards a production of anti-inflammatory cytokines could be a desirable effect to be evaluated in future drug studies of Linomide-like substances. At present, Linomide is not evaluable in MS clinical trials due to side-effects.     

Influence of IFN-β1b (Betaferon) on cytokine mRNA profiles in blood mononuclear cells and plasma levels of soluble VCAM-1 in multiple sclerosis

Inflammatory cell infiltration within the central nervous system (CNS) and upregulation of both pro- and anti-inflammatory cytokines are characteristic for multiple sclerosis (MS). Treatment with interferon-β1b (IFN-β1b) reduces the number and severity of MS relapses. To examine whether treatment with IFN-β1b affects levels of cytokine mRNA expressing blood mononuclear cells (MNC) we employed in-sit hybridization with synthetic oligonucleotide probes to detect and enumerate IFN-γ, TNF-α, IL-10, TGF-β and perforin mRNA expressing cells in MS patients before treatment with IFN-β1b and during tretmetn for 3–6 weeks and for 3–6 monts. Numbers of blood MNC spontaneously expressing TNF-α and IL-10 mRNA were lower after 3–6 months of treatment, while numbers of IFN-γ, TGF-β and perforin mRNA expressing MNC were not affected by treatment. IFN-β1b had no influence on levels of MBP-reactive IFN-γ, TNF-α, TGF-β, IL-10 or perforin mRNA expressing blood MNC determined after 3–6 weeks 3–6 months of treatment. Parallel measurements of plasma concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1) revealed elevated levels after 3–6 weeks of treatment and these levels remained higher after 3–6 months of treatment. The results suggest that IFN-β1b treatment upregulates plasma levels of sVCAM-1, but has little effects on numbers of blood MNC expressing mRNA of the pro- and anti-inflammatory cytokines under study.     

Local expression of cytokines in idiopathic inflammatory myopathies

H. Lepidi, V. Frances, D. Figarella-Branger, C. Bartoli, A. Machado-Baeta & J-F. Pellissier (1998) Neuropathology and Applied Biology , 24, 73–79
Local expression of cytokines in idiopathic inflammatory myopathies
The idiopathic inflammatory myopathies (IIM), including dermatomyositis (DM), polymyositis (PM), and inclusion body myositis (IBM), are regarded as autoimmune diseases. They are characterized by chronic lymphocytic and macrophagic infiltration in muscle tissue. Of particular importance in understanding the immune response to IIM is the specific pattern of locally produced cytokines. Frozen muscle tissues from IIM (5 DM, 3 PM, and 1 IBM) were used to investigate the cytokine responses. The RT-PCR technique was instrumental to determine the pattern of expression of pro-inflammatory (IL-1β, IL-6, TNF-α), Th1 (IFN-γ IL-2), and Th2 (IL-4) cytokines. Immunohistochemistry was also used to localize morphologically IFN-γ and IL-4. Our results show that pro-inflammatory cytokines and Th1 cytokines are mainly expressed in IIM. The accumulation of mononuclear inflammatory cells and the inflammatory syndrome in IIM are probably related in part to the production of pro-inflammatory cytokines. Moreover, the pattern of local cytokine expression is consistent with a Th1 immune response related to autoimmune diseases.     

SENSORY GANGLIONOPATHY ASSOCIATED WITH SJÖGREN'S SYNDROME PRESENTING WITH DYSPHAGIA

In immune-mediated demyelination of the nervous system, glial cell apoptosis has been observed recently; however, the relevance of the phenomenon and the characterization of the involved molecules are still controversial. Cytokines are secreted by many cells, including inflammatory and glial cells, and appear to play a relevant role in the peripheral nervous system (PNS) immuno-mediated demyelination, being active in promoting the damage to Schwann cells, myelin, and axons. Even though the exact role of the different cytokines is at present uncertain, they have a sequential different expression in PNS immune-mediated demyelination and could induce apoptotic death of Schwann cells in the vicinity of the inflammatory reaction via the expression of CD95 (Apo1/Fas). This study has been designed to detect in rat primary Schwann cell tissue cultures whether the administration of IL-1B and IFN-y can induce cell death. Identification of apoptotic Schwann cell was performed by morphological, immunohistochemical, and electron-microscopy analysis. Our results show that Schwann cells stimulated by proinflammatory cytokines IL-1B and IFN-y show morphological evidence of nuclear chromatin condesation at the DAPI staining and are TUNEL positive. The same features of apoptotic cell death were observed by electron microscopy. These findings provide evidence to support the hypothesis that cytokines can directly damage Schwann cells in disorders of the PNS.     

Cytokines Regulate L-Arginine-Dependent Cyclic GMP Production in Rat Glial Cells

We have previously demonstrated that primary astrocyte cultures from neonatal rat cortex and C6 glioma cells express a calcium-independent nitric oxide synthase (NOS) on induction with bacterial endotoxin (lipopolysaccharide, LPS). One hypothesis regarding the mechanism of the LPS induction is that it causes release of cytokines from these cells which then induce the enzyme directly. Such cytokine induction of NOS has been demonstrated in many extraneural cell types. l -Arginine-dependent increases in cyclic GMP correlate with smaller increases in accumulation of nitrite, the major oxidation product of nitric oxide, and hence can serve as a more sensitive measure of nitric oxide production. Here we provide evidence that interferon-γ (IFN-γ), interleukin (IL)-1β and tumour necrosis factor-α induce l -arginine-dependent cyclic GMP synthesis in C6 cells and that a combination of IFN-γ and IL-1β induce l -arginine-dependent cyclic GMP synthesis in astrocyte cultures, indicating that these cytokines induce NOS. In both cell types the induction by cytokines was less sensitive to inhibition by dexamethasone, IL-10 and IL-4 than was induction by LPS. These data suggest that cytokines can also induce a NOS in glial cells and that the mechanism of this induction may be more direct than that of LPS, since it is less sensitive to modulation by immunosuppressors. Due to the close associations of astrocytes with neurons and microvasculature, cytokine-induced NOS could have potentially important pathophysiological effects in the central nervous system.     

Increase of pro‐inflammatory cytokines in chronic sensory ataxic neuropathies

During cell‐mediated demyelination of the peripheral nervous system (PNS), pro‐inflammatory cytokines have a predictable pattern of expression and appear to be involved in damaging the myelin sheath and the axon. However, the role of these molecules regarding Schwann cell life and death is still controversial. In fact, some pro‐inflammatory cytokines act synergistically to kill PNS glial cells, but besides Schwann cell death they can induce cell proliferation, and both processes are related to cytokine dosage and time of exposure. In this preliminary study we stimulated Schwann cell cultures with 10 U/ml interleukin‐1 beta and 50 U/ml interferon‐gamma for 24, 48 and 72 hours. As previously reported we observed the peak of cell death and proliferation by 24 hours with both cytokines. At this time point, interestingly, in few Schwann cells belonging to the cytokine treated cultures and not to the untreated control cultures, we observed by electron microscopy analysis a cellular differentiation towards a myelin forming phenotype, even though our cultures were axon free as we have detected by immunocytochemical analysis with neurofilament antibody. In conclusion, we think that pro‐inflammatory cytokines besides promoting Schwann cell death and proliferation might be involved in the processes of cellular differentiation.     

Effect of high-dose methylprednisolone administration on immune functions in multiple sclerosis patients

Objectives – To investigate the in vivo effect of corticosteroid pulse therapy on immunocompetent cells in 18 patients given methylprednisolone to treat an acute episode of MS. Material and methods – Blood was sampled before and after 3 days of methylprednisolone administration at doses of 1 g/day. Lymphocyte subtyping was performed and whole blood cell cultures were used to measure the cytokine producing capacity for interleukin-1 (IL-1), interleukin-2 (IL-2), interferon-γ (IFN-γ), tumor necrosis factor-a (TNF-α) and interferon-α (IFN-α). In addition, serum levels of the immunoglobulin classes IgG, IgA and IgM were determined. Results – Before treatment, production of IL-1 was significantly increased in MS patients as compared to healthy controls. After therapy, production of all cytokines was significantly decreased, whereas there were significant increases in the numbers of monocytes, neutrophils and T and B lymphocytes. Treatment had no effect on serum immunoglobulin levels. Conclusion – An important mechanism for the antiinflammatory effect of corticosteroids in MS results from a suppression of the activation of the peripheral immune compartment through inhibition of cytokine production and lymphocyte endothelial adhesiveness.     

Comparative pharmacokinetics and pharmacodynamics of recombinant human interferon beta-1a after intramuscular and subcutaneous administration

The pharmacokinetics and pharmacodynamics of recombinant human interferon beta (IFN-β-la) were compared after intramuscular administration of two preparations (Rebif® and Avonex™) and subcutaneous administration of Rebif®. Healthy volunteers ( n = 30) received a single dose (6O μg) of each of the three treatments in a randomised crossover study. Serum concentrations of IFN-β were measured by enzyme-linked immunosorbent assay over 24 h after dosing. Pharmacodynamics were assessed by measurement of intracellular 2',5'-oligoadenylate synthetase activity, and serum neopterin and β₂-microglobulin concentrations, over 144 h after dosing. There was no significant difference between the three treatments in peak serum IFN-β concentrations ( C _max) or area under the concentration-time curve (AUC). No significant differences in pharmacodynamic measures were observed between the three treatments. It is concluded that the bioavailability of IFN-β-1a is equivalent after subcutaneous or intramuscular administration of Rebif®, and intramuscular administration of Avonex™.     

Antibodies to interleukin-1 inhibit cytokine-induced proliferation of neonatal rat Schwann cells in vitro

Unfractionated cytokines have been shown to induce in vitro proliferation of neonatal rat Schwann cells but the nature of the mitogen(s) is not known. A mixture of rabbit antibodies specific for recombinant interleukin-1α (IL-1α) and interleukin-1β (IL-1β) inhibited Schwann cell proliferation induced by unfractionated human cytokines whereas antibodies to interleukin-2 (IL-2) and control IgG did not. However, purified human IL-1 and recombinant human IL-1α or β did not induce Schwann cell proliferation on their own.     

AGEs对SH-SY5Y细胞增殖、凋亡以及阿尔茨海默病相关mRNA的影响

目的 研究晚期糖基化终产物(AGEs)对人神经母细胞瘤SH-SY5Y细胞株增殖、凋亡以及AD相关mRNA的影响。 方法 利用小牛血清白蛋白(BSA)和葡萄糖体外制备BSA-AGEs;将SH-SY5Y细胞与不同浓度BSA-AGEs保温后,MTT法测定SH-SY5Y细胞的增殖率,流式细胞仪测定细胞凋亡和细胞周期的变化,RT-PCR检测细胞中AD相关mRNA的表达水平。 结果 BSA-AGEs对SH-SY5Y细胞增殖有明显抑制作用,明显促进神经元的凋亡,细胞周期被阻滞于G1/G0期,呈药物浓度依赖性。RT-PCR结果表明,经过BSA-AGEs刺激后,IL-6、高迁移率族蛋白B1(HMGB1)、淀粉先质蛋白(APLP1) mRNA表达水平均明显升高。 结论 BSA-AGEs能有效抑制SH-SY5Y细胞的增殖,促进炎症细胞因子产生,诱导细胞凋亡,提示AGEs在AD的发生与发展过程中具有促进作用。     

ATP binding cassette transporter ABC1 is required for the release of interleukin-1beta by P2X7-stimulated and lipopolysaccharide-primed mouse Schwann cells

Schwann cells are best known as myelinating glial cells of the peripheral nervous system, but they also participate actively in the sphere of immunity by producing pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta). In a previous study, we demonstrated that posttranslational processing of IL-1beta by immune-challenged Schwann cells required the P2X7 receptor. Remarkably, the release of IL-1beta was not associated with cell death, indicating the involvement of an active mechanism. ATP binding cassette (ABC) transporters are known to transport leaderless secretory proteins, such as IL-1beta; therefore, we investigated whether such transporters were at work in Schwann cells. Mouse Schwann cells expressed ABC1 transporter mRNA and displayed the functional protein. Glybenclamide and diisothiocyanato-stilbene-disulfonic acid (DIDS), two blockers of chloride fluxes that drive the export activity of ABC1 transporters, inhibited IL-1beta release without altering its intracellular processing. Enhancing chloride efflux potentiated the release of IL-1beta, while decreasing it led to a strong reduction in its release. Because the stimulation of the P2X7 receptor also activates a chloride conductance, we investigated the possibility of a sole anionic pathway mobilized by the P2X7 receptor and ABC1. Glybenclamide and DIDS had no significant effects on the P2X7-activated chloride current suggesting therefore the existence of two different pathways. In summary, ABC1 transporters are required for the release of IL-1beta by mouse Schwann cells. Being associated together with chloride conductance, P2X7 receptors and ABC1 transporters delineate a subtle and complex regulation of IL-1beta production in mammalian Schwann cells. Furthermore, ABC1 transporters could be a target of therapeutic interest for regulating IL-1beta activity in neuroinflammation disorders.     

Involvement of γ and β Actin Isoforms in Mouse Neuroblastoma Differentiation

Two actin isoforms, γ and β, are contained within neuroblastoma cells. However, the relative amount and distribution of both isoforms within the cells are differentially regulated during neurite extension. The proportion of γ actin isoform became about four times greater than that of β actin during neuroblastoma cell differentiation. Additionally, whereas β actin appears to be concentrated in the cell cortex, γ actin is also present throughout the cell body. Upon differentiation, neuroblastoma cells reorganize their actin cytoskeleton and γ actin is induced to polymerize whereas β actin polymers are partially disassembled. Moreover, both actin isoforms are differentially distributed within differentiated cells. Thus, γ actin polymers are located both in the soma and proximal regions of extended neurites, whereas β actin is enriched in the terminal tip of the neurites. Our results strongly suggest that both actin isoforms are involved in a different way in neuroblastoma cell differentiation.     

Gabapentin Enhances the Morphine Anti-Nociceptive Effect in Neuropathic Pain via the Interleukin-10-Heme Oxygenase-1 Signalling Pathway in Rats

In the present study, we investigated the anti-inflammatory mechanisms by which gabapentin enhances morphine anti-nociceptive effect in neuropathic pain in rats and the interaction between the anti-nociceptive effects of gabapentin on morphine and the interleukin (IL)-10-heme-oxygenase (HO)-1 signal pathway in a rat model of neuropathic pain. The neuropathic pain model was induced via a left L5/6 spinal nerve ligation (SNL) in rats. The anti-nociceptive effect of gabapentin and IL-10 on morphine was examined over a 7-day period, and the effects of the anti-IL-10 and HO-1 inhibitor zinc protoporphyrin (ZnPP) on gabapentin/morphine co-injection were assessed. Drug administration was given over 7 days, and on day 8, both anti-inflammatory cytokine IL-10, a stress-induced protein HO-1 and pro-inflammatory cytokines IL-1β, IL-6 and TNF-α were measured. Gabapentin attenuated morphine tolerance over 7 days of co-administration, and reduced the expression of pro-inflammatory cytokines but increased IL-10 and HO-1 expression. The effect of gabapentin on morphine was partially blocked using the anti-IL-10 antibody or the HO-1 inhibitor zinc protoporphyrin. Our findings indicated that the anti-nociceptive effects of gabapentin on morphine might be caused by activation of the IL-10-HO-1 signalling pathway, which resulted in the inhibition of the expression of pro-inflammatory cytokines in neuropathic pain in the rat spinal cord.     

Tumor necrosis factor α and interleukin-6 mRNA expression in neonatal Lewis rat Schwann cells and a neonatal rat Schwann cell line following interferon γ stimulation

There is increasing evidence that Schwann cells play an important role in the pathogenesis of autoimmune inflammatory peripheral nerve disease. Schwann cells have been reported to express major histocompatibility complex class I and II (MHC I and II) and intercellular adhesion molecule-1 (ICAM-1), and to produce interleukin-1 (IL-1), prostaglandin E2 and thromboxane A2. In this study we investigated freshly dissociated neonatal Lewis rat Schwann cells and a SV40 transfected neonatal rat Schwann cell line (Schwann cell line) for production of mRNA for the immunomodulatory cytokines IL-2, IL-4, IL-6, IL-10, interferon-gamma (IFNγ), and tumor necrosis factor-alpha (TNFα) employing RT-PCR. Primary Schwann cells and Schwann cell line were examined following IFNγ stimulation and were found to express TNFα and IL-6 mRNA. These results further support a role for Schwann cell participation in inflammatory responses within the peripheral nervous system (PNS).     

Effects of interferon-γ, interleukin-1β, and tumor necrosis factor-α on the serotonin metabolism in the nucleus raphe dorsalis of the rat

Summary The effects of the cytokines interferon (IFN)-, interleukin (IL)-1, and tumor necrosis factor (TNF)- on the serotoninergic transmission in the nucleus raphe dorsalis (NRD) were studied after peripheral and central application. The studies were performed in the freely moving rat using differential pulse voltammetry with multicarbon fibre electrodes to study the extracellular levels of the serotonin (5-HT) metabolite 5-hydroxyindoleacetic acid (5-HIAA). The extracellular 5-HIAA levels were not changed in the NRD after peripheral application of rat recombinant IFN-, but elevated by the cytokines IL-1 and TNF-. After intracerebroventricular (i.c.v.) application the cytokines IFN-, IL-1 and TNF- stimulated the serotoninergic transmission in the NRD. Our data suggest that the effect of peripherally elevated cytokine concentrations on the serotonin metabolism in the NRD of the rat is cytokine-dependent. In this respect the T-cell and NK-cell cytokine IFN- acts clearly different when compared to the mainly macrophage-derived cytokines IL-1 and TNF-, and plays a different role in the communication between immune and central nervous system.     

Interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta inhibit cyclic AMP-induced Schwann cell differentiation.

Schwann cells differentiate in vivo in response to contact with axons, and cAMP simulates some of these aspects of differentiation in vitro, particularly morphologic changes and expression of certain phenotypic molecules. Unfractionated inflammatory cytokines inhibit cAMP-induced Schwann cell expression of galactolipids (Gal). We sought to identify which cytokines were responsible for this inhibition and to determine whether other phenotypic indicators of Schwann cell differentiation were also affected. Neonatal rat Schwann cells were incubated in vitro with 1 mM 8 Bromo cAMP (8 Br cAMP) with or without the addition of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or transforming growth factor-beta (TGF-beta). Cells were then examined for morphologic changes and for expression of surface Gal and low-affinity nerve growth factor receptor (NGFRp75), employing indirect immunofluorescence. 8 Br cAMP induced Schwann cell upregulation of Gal, downregulation of NGFRp75, and the cells became enlarged and somewhat amorphous and irregular in appearance. Cells treated with IFN-gamma or TNF-alpha alone were more bipolar and more evenly distributed on coverslips than were control cells, whereas TGF-beta alone induced elongated cells often in a swirling pattern. None of the cytokines alone induced upregulation of Gal or downregulation of NGFRp75. TNF-alpha, IFN-gamma, and TGF-beta inhibited the 8 Br cAMP-induced morphologic changes, as well as the upregulation of Gal and downregulation of NGFRp75. The other cytokines had no effects on Gal or NGFRp75 expression. Thus, these three cytokines, which are present in inflammatory lesions in the peripheral nervous system, are capable of inhibiting Schwann cell differentiation.