Differential induction of interleukin-12, interleukin-18, and interleukin-1β converting enzyme mRNA in experimental autoimmune encephalomyelitis of the Lewis rat

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Abstract

Experimental autoimmune encephalomyelitis (EAE) is a model of autoimmune central nervous system (CNS) disease that is mediated by autoreactive Th1 cells secreting the proinflammatory cytokine interferon (IFN)-γ. Interleukin (IL)-12 in its heterodimeric p35/p40 isoform and the recently described cytokine IL-18 potently induce T cell production of IFN-γ. Interleukin-1β converting enzyme (ICE) is required to convert IL-18 precursor protein into its biologically active mature form. In this study, we used semiquantitative reverse transciptase-polymerase chain reaction to determine steady state levels of IL-12, IL-18, and ICE mRNA in the spinal cord of Lewis rats at different stages of EAE. In control rats, we found significant IL-18, ICE, and IL-12p35, but not IL-12p40 mRNA expression. IL-18 mRNA increased during the acute stage of EAE together with a marked induction of ICE mRNA. IL-12p35 mRNA levels did not change significantly throughout the course of EAE. Surprisingly, the peak expression of IL-12p40 mRNA was delayed by several days relative to the peak of T cell infiltration and IFN-γ mRNA synthesis. Our data implicate the IL-18/ICE pathway in the amplification of Th1-mediated immune responses in the CNS but suggest a different, so far undefined role of endogenous IL-12 in the late effector phase of EAE.

Introduction

Experimental autoimmune encephalomyelitis (EAE) is a widely used model of autoimmune central nervous system (CNS) disease that mimics several aspects of human multiple sclerosis (Raine, 1984; Wekerle, 1993). EAE is mediated by autoreactive Th1 cells secreting interferon (IFN)-γ and interleukin (IL)-2. For the generation and efficient activation of Th1 cells several costimulatory signals have to be delivered during initial stages of a cellular immune response (Abbas et al., 1996). IL-12 is a heterodimeric cytokine that is composed of two different covalently linked subunits, p35 and p40. Its production by accessory cells during the initiation of an immune response polarizes naive T cells toward a Th1 pattern of cytokine production (Trinchieri, 1993). IL-18 was discovered only recently as a `IFN-γ-inducing-factor' in a mouse model of endotoxic shock (Okamura et al., 1995). IL-18 is synthesized as an inactive precursor protein that exhibits structural homology to IL-1 (Bazan et al., 1996) and accordingly has to be processed by the caspase IL-1β converting enzyme (ICE) to get functionally active (Ghayur et al., 1997; Gu et al., 1997). Similar to IL-12, IL-18 enhances the production of IFN-γ by Th1 cells although both cytokines apparently use different intracellular signaling pathways (Jacobson et al., 1995; Thierfelder et al., 1996; Kohno et al., 1997; Matsumoto et al., 1997; Robinson et al., 1997).

In line with a Th1-mediated immunopathogenesis, IL12p40 mRNA has been detected in active multiple sclerosis lesions (Windhagen et al., 1995) and disease-enhancing effects of IL-12 have been demonstrated in EAE (Leonard et al., 1995; Segal and Shevach, 1996; Smith et al., 1997; Segal et al., 1998). IL-18 mRNA has been found in active stages of murine autoimmune diabetes (Rothe et al., 1997) but its expression in CNS autoimmune diseases has not been studied so far. Accordingly, the relative contribution of both cytokines to the regulation of Th1-mediated immune responses in the CNS is currently unknown. In the present study we used a semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) approach to analyze the time course of IL-12 p35/p40, IL-18, and ICE mRNA expression in the spinal cord of Lewis rats at different stages of EAE actively induced with the encephalitogenic 68–86 peptide of guinea pig myelin basic protein.

Section snippets

Animals

EAE was induced in 10 to 14 weeks old female Lewis rats (Charles River Laboratories, Kisslegg, Germany) by immunization with a synthetic peptide (YGSLPQKSQRSQDENPV; 25 μg per animal) corresponding to the amino acids 68–86 (Fritz and McFarlin, 1989) of guinea pig myelin basic protein (MBP68–86) according to standard procedures (Swanborg, 1988). High performance liquid chromatography-purified MBP68–86 peptide was purchased from the Department of Biochemistry, Georg-August-University, Göttingen,

Clinical course of EAE

MBP68–86-immunized Lewis rats exhibited a paresis of the tail as the first clinical sign of EAE at day 11 (Fig. 3). Thereafter, the animals developed a progressive paraparesis which usually peaked at day 15. During the following days, the rats recovered gradually. On day 21, all animals were asymptomatic. With the currently used immunization regime the manifestation rate of EAE was 100%.

RT-PCR analysis of cytokine, ICE, and CD3 mRNA levels

In untreated control animals, we found significant expression of IL-18, IL-12p35, and ICE mRNA (Fig. 2Fig. 3

Discussion

In the present study we used semiquantitative RT-PCR analysis of steady state mRNA levels to characterize the expression of two IFN-γ-inducing cytokines, IL-18 and IL-12, during the course of EAE in Lewis rats. In addition, we analyzed the time course of ICE mRNA expression since this enzyme has been shown to process IL-18 precursor protein into its functionally active mature form. As main finding we show a coincident induction of IL-18 and ICE mRNA that peaked during the active disease stage

Acknowledgements

We thank B. Blomenkamp for excellent technical assistance and U. Pippirs for help with automated DNA sequencing. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 194, B6). G.S. holds a Hermann-and-Lilly-Schilling professorship.

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