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
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.
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