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

doi:10.1016/S0165-5728(96)00131-2

Journal of Neuroimmunology

Volume 71, Issues 1–2, December 1996, Pages 65-71

https://doi.org/10.1016/S0165-5728(96)00131-2 Get rights and content

Abstract

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).

Introduction

Guillain-Barré syndrome (GBS) and chronic idiopathic demyelinating polyneuropathy (CIDP) are autoimmune diseases of peripheral nerve and characterised by demyelination (England, 1990) and secondary axonal degeneration (Berciano et al., 1993; Dyck, 1993). Schwann cells, the myelin forming cells of the peripheral nervous system have also been shown to express MHC II in studies of GBS and CIDP nerve (Pollard et al., 1986; Pollard et al., 1987) and on Schwann cells in vitro after exposure to the cytokines IFNγ and/or TNFα, and activated CD4⁺ T-cells (Samuel et al., 1987; Kingston et al., 1989; Argall et al., 1992). In line with MHC II expression there is also evidence that Schwann cells can act as non-professional antigen presenting cells (Wekerle et al., 1986; Argall et al., 1992).

Induction of MHC II expression and the finding that Schwann cells can produce IL-1 (Bergsteindottir et al., 1991) furthers the possibility that the Schwann cells may be more than a passive target and may play an active role in immune responses. As IL-1 is an inducer of IL-2 (Smith et al., 1980), it is relevant that serum from GBS patients shows increased level of IL-2 and IL-2 receptor (Hartung et al., 1990; Hartung et al., 1991; Hartung and Toyka, 1990) with a decrease in parallel with recovery (Hartung et al., 1991; Bansil et al., 1991; Bansil et al., 1992). Further, the levels of TNFα and IL-6 in GBS patient sera also correlates with disease severity (Weller et al., 1991; Sharief et al., 1993; Shimada et al., 1993). We therefore examined the further possibility that Schwann cells may be able to produce upregulatory cytokines mRNA for IL-2, IL-6, IFNγ, TNFα as well as IL-4 and IL-10. The last two cytokines are important mediators in immune suppression (Racke et al., 1994; Rott et al., 1994; Issazadeh et al., 1995; Sugiyama et al., 1995; Jander et al., 1996). We employed freshly dissociated neonatal rat Schwann cells and a neonatal rat Schwann cell line (Tennekoon, 1987; Schneider-Schaulies et al., 1991).

Section snippets

SV-40 transfected neonatal rat Schwann cell line (Schwann cell line)

To minimise use of neonatal rats and to provide a system for optimising expression of cytokine mRNA, the Schwann cell line (MT4SV-H1) (Tennekoon, 1987) kindly provided by Dr. J. Archelos (Schneider-Schaulies et al., 1991) was employed. The cells were cultured in 25 cm² tissue culture flasks (Falcon) in 5 ml EMEM (Sigma) containing glucose, 10% FCS (Trace Bioscientific), 50 U/ml penicillin G (Trace Bioscientific), 50 mg/ml streptomycin sulphate (Trace Bioscientific), and 200 mM L-glutamine

TNFα and IL-6 mRNA expression in the Schwann cell line

No TNFα mRNA expression was detected following 20 IU IFNγ stimulation (Fig. 2). However, TNFα mRNA expression from cultures stimulated with 100 or 400 IU IFNγ was detected by 12 h stimulation and persisted up to 48 h (Fig. 3Fig. 4, lane 7a, 14a, 7b). Expression of IL-6 mRNA following 100 IU or 400 IU IFNγ stimulation was first detected by 12 h (Fig. 3a, lane 4 and Fig. 4a, lane 4 respectively) and still present after 48 h (Fig. 3b lane 11, and Fig. 4b, lane 11), while IL-6 expression following

Discussion

Our results show for the first time that the SV40 neonatal rat Schwann cell line and inbred Lewis rat primary Schwann cells in vitro express mRNA for TNFα and IL-6 following IFNγ stimulation. The time at which mRNA was detectable depended on the IFNγ concentration. In the Schwann cell line stimulated with 20 IU IFNγ, TNFα mRNA was undetectable at all time tested, and IL-6 mRNA was detectable after 48 h (Fig. 3). However, following 100 and 400 IU IFNγ stimulation, TNFα and IL-6 transcripts were

Acknowledgements

The authors particularly thank Dr. Marianne Frommer and Dr. Neville Firth (School of Biological Science, Laboratory of Molecular Genetics, The University of Sydney), for discussions regarding PCR techniques. We thank Mr. James Bonner for preparing activated T-cells. This study was supported by The Australian Agency for International Development and National Health and Medical Research Council of Australia.

References (44)

K.G Argall et al.
Interactions between CD4⁺ T-cells and rat Schwann cells in vitro. 1. Antigen presentation by Lewis rat Schwann cells to P2-specific CD4⁺ T-cell lines
J. Neuroimmunol.
(1992)
E.N Benveniste et al.
Induction and regulation of interleukin-6 gene expression in rat astrocytes
J. Neuroimmunol.
(1990)
J Brockes et al.
Studies on cultured rat Schwann cellsI. Establishment of purified populations from cultures of peripheral nerve
Brain Res.
(1979)
A.L Constable et al.
Production of the prostanoids, prostaglandin E2 and thromboxane B2 by rat Schwann cells in vitro
Brain Res.
(1994)
K Fields et al.
A monoclonal antibody equivalent to anti-rat neural antigen as a marker for Schwann cells
Neurosci.
(1985)
R Gold et al.
Synergistic effect of IFNγ and TNFα on expression of immune molecules and antigen presentation by Schwann cells
Cell. Immunol.
(1995)
S Issazadeh et al.
Cytokine production in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis: dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin, tumor necrosis factor α and tumor necrosis factor β
J. Neuroimmunol.
(1995)
J.D Pollard et al.
Class II antigen expression and T lymphocyte subsets in chronic inflammatory demyelinating polyneuropathy
J. Neuroimmunol.
(1986)
J Schneider-Schaulies et al.
Down-regulation of myelin associated glycoprotein on Schwann cells by interferon-gamma and tumor necrosis factor-alpha affects neurite outgrowth
Neuron
(1991)
M Weller et al.
Comparative analysis of cytokine patterns in immunological, infectious, and oncological neurological disorders
J. Neurol. Sci.
(1991)

F Aloisi et al.

Production of hemolymphopoietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-1 beta and tumor necrosis factor-alpha

J. Immunol.

(1992)

P.J Armati et al.

Rat and human Schwann cells in vitro can synthesize and express MHC molecules

Muscle Nerve

(1990)

S Bansil et al.

Clinical correlation with serum-soluble interleukin-2 receptor levels in Guillain-Barré syndrome

Neurol.

(1991)

S Bansil et al.

Elevated neopterin levels in Guillain-Barré syndrome. Further evidence of immune activation

Arch. Neurol.

(1992)

J Berciano et al.

Axonal form of Guillain-Barré Syndrome

Muscle Nerve

(1993)

K Bergsteindottir et al.

Rat Schwann cells produce interleukin-1

J. Neuroimmunol.

(1991)

K Bergsteindottir et al.

Rat Schwann cell can be induced to express major histocompatibility complex class II molecules in vivo

J. Neurocytol.

(1992)

L.M Bolin et al.

Interleukin-6 production by Schwann cells and induction in sciatic nerve injury

J. Neurochem.

(1995)

B.I.Y Chung et al.

Differential tumor necrosis factor a expression by astrocytes from experimental allergic encephalomyelitis-susceptible and -resistant rat strains

J. Exp. Med.

(1991)

P Dyck

Is there an axonal variety of GBS?

Neurol.

(1993)

J.D England

Guillain-Barré Syndrome

Ann. Rev. Med.

(1990)

K Frei et al.

On the cellular source and function of interleukin-6 produced in the central nervous system in viral diseases

Eur. J. Immunol.

(1989)

Cited by (48)

Upregulation of neuronal nitric oxide synthase in the periphery promotes pain hypersensitivity after peripheral nerve injury
2011, Neuroscience
Citation Excerpt :
Moreover, total levels of nNOS protein and transcripts were increased in the ipsilateral sciatic nerve following nerve injury (Fig. 6A, B). During Wallerian degeneration following nerve injury, Schwann cells enveloping degenerating axons undergo marked reactive changes, begin to phagocytose myelin debris, and synthesize several biological molecules, including nerve growth factor, tumor necrosis factor-α, IL-1β, and IL-6 (Matsuoka et al., 1991; Murwani et al., 1996; Shamash et al., 2002). In this study, we could not investigate the origin or mechanism of the nNOS induction in the sciatic nerve, but it may result from the activation of a specific receptor in Schwann cells leading to modulation of nNOS expression via a transcription pathway such as that of c-Jun N-terminal kinase (JNK) or p38 kinase, or increased synthesis of a biological molecule in Schwann cells following nerve injury that modulates nNOS expression via interaction with proinflammatory cytokines or chemokines (Oh et al., 2001; Scholz and Woolf, 2007).
Peripheral nerve injury often results in neuropathic pain that is manifested as hyperalgesia, and allodynia. Several studies suggest a functional role for neuronal nitric oxide synthase (nNOS) in the development or maintenance of neuropathic pain, but such a contribution remains unclear. In our current study, we found that intraplantar injection of the NOS substrate l-arginine or NO donor 3-morpholino-synonimine (SIN-1) produced mechanical hypersensitivity that lasted more than 24 h. Following L5 spinal nerve ligation (L5 SNL), immunoreactivity for nNOS in the ipsilateral L5 but not L4 dorsal root ganglion (DRG) was dramatically increased in mainly small- and medium-sized neurons and non-neuronal cells. L5 SNL caused increased nNOS immunoreactivity in the ipsilateral sciatic nerve, mainly in Schwann cells and the ipsilateral glabrous hind paw skin, mainly on the basement membrane. Furthermore, total nNOS protein and mRNA in the ipsilateral sciatic nerve and hind paw skin were markedly upregulated following nerve injury. Intraplantar injection of the NOS inhibitor 7-nitroindazole (7-NI) or the non-specific NOS inhibitor l-N^G-nitro-arginine methyl ester (l-NAME) effectively suppressed SNL-induced mechanical allodynia. Collectively, these data suggest that in the periphery nNOS upregulation induced by peripheral nerve injury contributes to mechanical hypersensitivity during the maintenance phase of neuropathic pain. Blocking nNOS signaling in the periphery may thus be a novel therapeutic strategy for the treatment of neuropathic pain.
The neuro-immune balance in neuropathic pain: Involvement of inflammatory immune cells, immune-like glial cells and cytokines
2010, Journal of Neuroimmunology
Citation Excerpt :
As a result of compromised myelin integrity of Aβ afferents, these normally mechanosensitive fibres promote mechanical nociception (Devor, 2006). Additionally within hours of the peripheral nerve injury activated Schwann cells begin to secrete other mediators including; pro-inflammatory cytokines, TNF (Murwani et al., 1996; Wagner and Myers, 1996), IL-1β (Bergsteinsdottir et al., 1991; Shamash et al., 2002) and IL-6 (Bolin et al., 1995); PGE2 (Muja and DeVries, 2004); ATP (Liu et al., 2005); and chemokines, such as leukemia inhibitory factor (LIF) and MCP-1, which promote continued neuroinflammation through recruitment of macrophages (Tofaris et al., 2002). TNF and IL-1β can directly stimulate neuronal hyperexcitability of A- and C-fibres (see Section 5.1 for more details, Schafers and Sorkin, 2008), whilst ATP can sensitise nociceptors and increase axonal excitability in peripheral nerves (Moalem et al., 2005).
In a large proportion of individuals nervous system damage may lead to a debilitating chronic neuropathic pain. Such pain may now be considered a neuro-immune disorder, since recent data indicate a critical involvement of innate and adaptive immune responses following nerve injury. Activation of immune and immune-like glial cells in the injured nerve, dorsal root ganglia and spinal cord results in the release of both pro- and anti-inflammatory cytokines, as well as algesic and analgesic mediators, the balance of which determines whether pain chronicity is established. This review will critically examine the role of the immune system in modulating chronic pain in animal models of nervous system injury, and highlight the possible therapeutic opportunities to intervene in the development and maintenance of neuropathic pain.
Frataxin deficiency induces Schwann cell inflammation and death
2009, Biochimica et Biophysica Acta - Molecular Basis of Disease
Mutations in the frataxin gene cause dorsal root ganglion demyelination and neurodegeneration, which leads to Friedreich's ataxia. However the consequences of frataxin depletion have not been measured in dorsal root ganglia or Schwann cells. We knocked down frataxin in several neural cell lines, including two dorsal root ganglia neural lines, 2 neuronal lines, a human oligodendroglial line (HOG) and multiple Schwann cell lines and measured cell death and proliferation. Only Schwann cells demonstrated a significant decrease in viability. In addition to the death of Schwann cells, frataxin decreased proliferation in Schwann, oligodendroglia, and slightly in one neural cell line. Thus the most severe effects of frataxin deficiency were on Schwann cells, which enwrap dorsal root ganglia neurons. Microarray of frataxin-deficient Schwann cells demonstrated strong activations of inflammatory and cell death genes including interleukin-6 and Tumor Necrosis Factor which were confirmed at the mRNA and protein levels. Frataxin knockdown in Schwann cells also specifically induced inflammatory arachidonate metabolites. Anti-inflammatory and anti-apoptotic drugs significantly rescued frataxin-dependent Schwann cell toxicity. Thus, frataxin deficiency triggers inflammatory changes and death of Schwann cells that is inhibitable by inflammatory and anti-apoptotic drugs.
Localisation and modulation of prostanoid receptors EP1 and EP4 in the rat chronic constriction injury model of neuropathic pain
2007, European Journal of Pain
Citation Excerpt :
We have shown that Schwann cells express EP1 and EP4 prostaglandin receptors, in both intact and neuropathic peripheral nerves, although it did not prove possible to assess by immunofluorescence whether or not this was altered following CCI. A possible role for Schwann cells in inflammatory responses has been suggested from studies on cultured cells (Constable et al., 1994; Bolin et al., 1995; Murwani et al., 1996; Muja et al., 2001), but relatively little data has hitherto been available from in vivo studies. A recent study in the Chung L5 spinal root injury model (Takahashi et al., 2004) showed a very early elevation of COX-2 immunoreactivity in S-100 positive Schwann cells at 1–3 days post-injury, the number of S-100 positive cells themselves being unaltered.
Immunohistochemistry was used to examine the expression of prostaglandin E₂ receptors EP1 and EP4 in sciatic nerves from the rat chronic constriction injury (CCI) model of neuropathic pain. At 21 days post-surgery the CCI rats had developed mechanical hyperalgesia on the operated side, and quantitative image analysis showed a highly significant doubling of the area occupied by EP1- and EP4-positive pixels in sections from CCI nerves when compared to sham-operated controls. Co-localisation studies with the marker ED1 revealed that 73% of the EP1-positive cells and 54% of the EP4-positive cells in the injured nerves represented infiltrating macrophages. Cells negative for ED1 and positive for either EP1 or EP4 were characterised as Schwann cells from their morphology and expression of myelin basic protein and S100 antigens. Similar EP1- and EP4-positive Schwann cell profiles were observed in sections of uninjured control nerves. Low levels of EP receptor expression were found in neurofilament-immunostained axons, but no consistent differences were observed in the levels of axonal EP staining between CCI and control tissue. These data provide further evidence of the importance of prostaglandins in the pathogenesis of neuropathic pain, and suggest that not only infiltrating macrophages but also Schwann cells may be involved in the modulation of these mediators in response to nerve injury.
Immune and inflammatory mechanisms in neuropathic pain
2006, Brain Research Reviews
Tissue damage, inflammation or injury of the nervous system may result in chronic neuropathic pain characterised by increased sensitivity to painful stimuli (hyperalgesia), the perception of innocuous stimuli as painful (allodynia) and spontaneous pain. Neuropathic pain has been described in about 1% of the US population, is often severely debilitating and largely resistant to treatment. Animal models of peripheral neuropathic pain are now available in which the mechanisms underlying hyperalgesia and allodynia due to nerve injury or nerve inflammation can be analysed. Recently, it has become clear that inflammatory and immune mechanisms both in the periphery and the central nervous system play an important role in neuropathic pain. Infiltration of inflammatory cells, as well as activation of resident immune cells in response to nervous system damage, leads to subsequent production and secretion of various inflammatory mediators. These mediators promote neuroimmune activation and can sensitise primary afferent neurones and contribute to pain hypersensitivity. Inflammatory cells such as mast cells, neutrophils, macrophages and T lymphocytes have all been implicated, as have immune-like glial cells such as microglia and astrocytes. In addition, the immune response plays an important role in demyelinating neuropathies such as multiple sclerosis (MS), in which pain is a common symptom, and an animal model of MS-related pain has recently been demonstrated. Here, we will briefly review some of the milestones in research that have led to an increased awareness of the contribution of immune and inflammatory systems to neuropathic pain and then review in more detail the role of immune cells and inflammatory mediators.
Oxidative Stress and Excitatory Neurotoxins in Neuropathy
2005, Peripheral Neuropathy: 2-Volume Set with Expert Consult Basic

View all citing articles on Scopus

View full text

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

Abstract

Introduction

Section snippets

SV-40 transfected neonatal rat Schwann cell line (Schwann cell line)

TNFα and IL-6 mRNA expression in the Schwann cell line

Discussion

Acknowledgements

J. Neuroimmunol.

J. Neuroimmunol.

Brain Res.

Brain Res.

Neurosci.

Cell. Immunol.

J. Neuroimmunol.

J. Neuroimmunol.

Neuron

J. Neurol. Sci.

Production of hemolymphopoietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-1 beta and tumor necrosis factor-alpha

J. Immunol.

Rat and human Schwann cells in vitro can synthesize and express MHC molecules

Muscle Nerve

Clinical correlation with serum-soluble interleukin-2 receptor levels in Guillain-Barré syndrome

Neurol.

Elevated neopterin levels in Guillain-Barré syndrome. Further evidence of immune activation

Arch. Neurol.

Axonal form of Guillain-Barré Syndrome

Muscle Nerve

Rat Schwann cells produce interleukin-1

J. Neuroimmunol.

Rat Schwann cell can be induced to express major histocompatibility complex class II molecules in vivo

J. Neurocytol.

Interleukin-6 production by Schwann cells and induction in sciatic nerve injury

J. Neurochem.

Differential tumor necrosis factor a expression by astrocytes from experimental allergic encephalomyelitis-susceptible and -resistant rat strains

J. Exp. Med.

Is there an axonal variety of GBS?

Neurol.

Guillain-Barré Syndrome

Ann. Rev. Med.

On the cellular source and function of interleukin-6 produced in the central nervous system in viral diseases

Eur. J. Immunol.