- Email: [email protected]
Interleukin-18 is induced in acute inflammatory demyelinating polyneuropathy
Journal of Neuroimmunology 114 (2001) 253–258 www.elsevier.com / locate / jneuroin
Interleukin-18 is induced in acute inflammatory demyelinating polyneuropathy Sebastian Jander*, Guido Stoll ¨ , Germany Department of Neurology, Heinrich-Heine-University, Moorenstraße 5, D-40225 Dusseldorf Received 20 September 2000; received in revised form 5 December 2000; accepted 12 December 2000
Abstract T lymphocytes of the Th1 subset producing the proinflammatory cytokine interferon-g (IFN-g) have been implicated in the pathogenesis of immune-mediated diseases of the peripheral nervous system (PNS) such as the acute Guillain–Barre´ syndrome (GBS) and its animal model experimental autoimmune neuritis (EAN). Interleukin-18 (IL-18) is a potent IFN-g-inducing cytokine that is synthesized as an inactive precursor molecule and cleaved by caspase-1 into its mature active form. In our present study we analyzed the expression of IL-18 and caspase-1 in the nerve roots of EAN rats using reverse transcriptase–polymerase chain reaction and immunocytochemistry. Using an enzyme-linked immunosorbent assay, we furthermore determined IL-18 protein levels in paired serum and cerebrospinal fluid (CSF) samples from patients with GBS as well as from noninflammatory neurologic disease (NIND) controls. In EAN, IL-18 and caspase-1 mRNA levels in the nerve roots increased during the stage of active disease progression. Immunocytochemically, both perivascular and parenchymal IL-18 protein expression was increased in the roots of EAN rats and mainly associated with ED11 macrophages stained on serial sections. IL-18 serum levels were significantly higher in GBS patients than in NIND controls (238671 vs. 4267 pg / ml, P,0.001). Our data implicate the Th1-inducing cytokine IL-18 in the pathogenesis of acute immune-mediated PNS demyelination. 2001 Elsevier Science B.V. All rights reserved. Keywords: Peripheral nervous system; Cytokine; Demyelination; Interleukin-18; Interferon-g; Caspase-1; Th1 cells
1. Introduction The Guillain–Barre´ syndrome (GBS) is an acute inflammatory demyelinating polyneuropathy that frequently occurs as a sequela of gastrointestinal infections and leads to progressive ascending tetraparesis (Hughes et al., 1999). Several aspects of the disease are mimicked in the model of experimental autoimmune neuritis (EAN) which can be induced in susceptible rat strains by sensitization against peripheral nervous system (PNS) myelin constituents such as the P2 protein (Hartung and Toyka, 1990). Pathophysiological studies have suggested that T helper cell-dependent activation of macrophages is the principal mechanism underlying immune-mediated myelin destruction in GBS and EAN (Hartung and Toyka, 1990; Stoll and Hartung, 1992). Depending on their specific pattern of cytokine production T helper cells can be subdivided into
*Corresponding author. Tel.: 149-211-81-18978; fax: 149-211-8118485. E-mail address: [email protected] (S. Jander).
two major subpopulations, i.e. Th1 cells secreting the proinflammatory cytokines interferon (IFN)-g and tumor necrosis factor-a and Th2 cells secreting antiinflammatory cytokines such as interleukin (IL)-4 and IL-10 (Abbas et al., 1996). In EAN, T cells infiltrating nerve roots abundantly express IFN-g mRNA and protein (Gillen et al., 1998; Schmidt et al., 1992). Therefore, it is a widely accepted view that disease induction in the EAN model as well as in human GBS follows a Th1-dependent immunopathogenesis (Zhu et al., 1998). The generation and activation of Th1 cells depends on the appropriate delivery of specific costimulatory signals during initial stages of a cellular immune response (Abbas et al., 1996). Antigen-presenting cells such as activated macrophages produce the immunoregulatory cytokines IL12 and IL-18 which are potent IFN-g-inducing factors and thereby favor a Th1-like polarization of T cell-mediated immune responses (Trinchieri, 1993; Kohno and Kurimoto, 1998). IL-18 was discovered only recently in a mouse model of endotoxic liver injury (Okamura et al., 1995). IL-18 is synthesized as an inactive precursor molecule that exhibits structural homology to IL-1b
0165-5728 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0165-5728( 00 )00460-4
254
S. Jander, G. Stoll / Journal of Neuroimmunology 114 (2001) 253 – 258
(Bazan et al., 1996) and accordingly has to be cleaved by caspase-1 (previously termed IL-1b converting enzyme) into its mature active form (Ghayur et al., 1997; Gu et al., 1997). In experimental autoimmune encephalomyelitis (EAE) a coordinated increase of both IL-18 and caspase-1 mRNA occurs during active disease progression (Jander and Stoll, 1998) suggesting a crucial role of this cytokine pathway in disease induction. So far, the mechanisms initiating the Th1-mediated immune response in acute inflammatory polyneuropathies are largely unknown. In our present study we have used reverse transcriptase–polymerase chain reaction (RT– PCR) and immunocytochemistry to analyze the expression of IL-18 and caspase-1 in nerve roots during the course of actively induced EAN in Lewis rats. To further substantiate a possible role of IL-18 in the pathogenesis of human PNS disease we have additionally measured serum and cerebrospinal fluid (CSF) levels of IL-18 in patients with GBS.
2. Methods
2.1. Animal experiments EAN was induced in 8-week-old female Lewis rats by immunization with bovine PNS myelin as described previously (Gillen et al., 1998). Disease severity was scored as follows: 0, no clinical signs; 0.5, loss of tail tone; 1, complete tail paresis; 2, incomplete hind limb paresis; 3, complete paraplegia. For RT–PCR analysis, EAN animals along with normal controls (n53–4 in each group) were sacrificed on days 11, 13, 15, 21, and 28 by an overdose of ether. Nerve roots were prepared rapidly, snap frozen in liquid nitrogen, and stored at 2808C until RNA preparation. For immunocytochemistry, additional rats (n53 in each group) were perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The lumbosacral spinal cord with adhering nerve roots was prepared, postfixed overnight in the same fixative, and embedded into paraffin.
2.2. RT–PCR Total RNA was isolated from the nerve roots according to standard procedures (Chomczynski and Sacchi, 1987) and reverse transcribed into cDNA as detailed elsewhere (Gillen et al., 1998). cDNA equivalent to 20 ng of total RNA was subjected to subsequent PCR analysis using primers specific for IL-18, caspase-1, or the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as described previously (Jander and Stoll, 1998). In preliminary experiments, optimal cycling con-
ditions were established allowing amplification of each cDNA in the linear range. Cycle numbers were as follows: IL-18528; caspase-1529; and GAPDH520. PCR products were separated on 1.5% agarose gels containing 10 mg / ml ethidium bromide, photographed using a CSCcamera (Cybertech, Berlin, Germany) and densitometric analysis was performed with TINA 2.1 software (Raytest, Straubenhardt, Germany).
2.3. Immunocytochemistry Longitudinal 5 mm sections of paraffin-embedded nerve roots were deparaffinized in xylene and rehydrated in a descending series of ethanol. After blocking of nonspecific binding sites with 3% normal horse serum, goat anti-rat IL-18 primary antibody (R&D Systems, Minneapolis, MN) at 2 mg / ml final working concentration was incubated overnight at 48C. Detection was performed with biotinylated horse anti-goat secondary antibody (Vector Laboratories, Burlingame, CA) and the ABC Elite kit (Vector) with diaminobenzidine as substrate. For cellular identification, adjacent serial sections were incubated with mAb ED1 against phagocytic macrophages (Serotec, Oxford, UK) at 1:2000 dilution, followed by biotinylated horse anti-mouse secondary antibody (Vector) and detection reagents as above. As control, replacement of primary antibodies with irrelevant goat or mouse IgG led to a disappearance of immunostaining.
2.4. Patients A total of 36 individuals were studied. Paired serum and CSF samples were collected between 1995 and 1999 and stored at 2708C. Before freezing, CSF samples were centrifuged for 5 min at high speed to remove cells. Eighteen patients met the established diagnostic criteria for acute GBS. All had rapidly progressive motor weakness of lower and upper extremities, areflexia, and mild sensory signs. Routine CSF studies showed normal cell counts in all patients. CSF protein content was elevated in 13, but normal in five subjects. Electrophysiologic measurements suggested generalized or focal demyelination in 15 cases, while predominantly axonal affection was found in three patients. As a control group [noninflammatory neurologic diseases (NIND)] 18 patients with one of the following diagnoses were studied: tension headache, depression, normal pressure hydrocephalus, migraine, disc herniation, pseudotumor cerebri, ischemic stroke, epilepsy, subcortical arteriosclerotic encephalopathy, Alzheimer’s disease. In all NIND patients, routine CSF analysis was normal and review of the clinical data revealed no evidence for systemic or localized inflammatory, infectious, or malignant disease.
S. Jander, G. Stoll / Journal of Neuroimmunology 114 (2001) 253 – 258
2.5. Enzyme-linked immunosorbent assay ( ELISA)
3. Results
The QuantikineE human IL-18 ELISA kit (R&D Systems, Minneapolis, MN) was used according to the manufacturer’s instructions. Both serum and CSF samples were tested at 1:2 dilution. To correct CSF IL-18 levels for blood CSF-barrier dysfunction typical in GBS we calculated IL-18 index values as follows: [IL-18 CSF / IL-18 serum :albumin CSF / albumin serum ].
3.1. Expression of IL-18 and caspase-1 mRNA during EAN
For statistical analysis, Mann–Whitney U-test was performed using GraphPad Prism 3.00 software (GraphPad Software, San Diego, CA). P-values,0.05 were considered as statistically significant.
255
Following immunization with bovine PNS myelin the animals developed tail paralysis as the first sign of EAN at days 10–11 after immunization, progressed until day 15 when complete paraparesis of the hind limbs was reached, and recovered spontaneously thereafter (Fig. 1A). On day 28, all animals were asymptomatic. In the nerve roots of normal control rats low constitutive levels of both IL-18 and caspase-1 mRNA were detected. Both mRNAs increased above baseline levels at the onset
Fig. 1. (A) Clinical course of actively induced EAN in Lewis rats. (B,C) RT–PCR analysis of IL-18 and caspase-1 mRNA levels in nerve roots during the course of actively induced EAN in Lewis rats. (B) Representative original RT–PCR findings. GAPDH was used as ‘house-keeping gene’ control in subsequent semiquantitative analysis. (C) Levels of IL-18 and caspase-1 mRNA relative to those of the ‘house-keeping gene’ GAPDH in n53–4 animals on each time point. Bars denote mean6S.E.M.
256
S. Jander, G. Stoll / Journal of Neuroimmunology 114 (2001) 253 – 258
of clinical disease at day 11 after immunization (Fig. 1B,C). IL-18 mRNA levels continued to increase until day 13 and showed a protracted decrease thereafter. Levels of caspase-1 mRNA decreased somewhat more rapidly already at day 13 after immunization. However, both messages remained elevated above baseline levels until day 28.
3.2. Immunocytochemical localization of IL-18 protein in inflamed nerve roots To corroborate our mRNA findings at the protein level we performed immunocytochemical experiments using an affinity-purified polyclonal antibody against rat IL-18. In
control nerves, weak constitutive expression on longitudinally orientated cells resembling resident PNS macrophages was found. In line with the PCR data a clear increase of IL-18 immunoreactivity occurred at days 11 and 13 after immunization with a subsequent decline beyond day 15 (Fig. 2A–D). At high magnification, IL-18 induction appeared to be mainly associated with perivascular infiltrates (Fig. 2B), but was also observed on parenchymal cells (Fig. 2D). At both sites, staining of serial sections revealed extensive colocalization of IL-18 immunoreactivity with the phagocyte marker ED1 (Fig. 2A,C) suggesting that IL-18 in EAN roots was mainly expressed by activated macrophages.
Fig. 2. Immunocytochemical localization of IL-18 protein on longitudinal paraffin sections of EAN roots on day 11 after immunization. A,B as well as C,D are taken from pairs of adjacent serial sections stained for the macrophage marker ED1 (A,C) and IL-18 (B,D), respectively. Extensive colocalization between both antigens is evident in perivascular infiltrates (A,B) as well as parenchymal cells (C,D). Arrowheads in C and D point to cells that coexpress IL-18 and ED1 antigen. Bars: 25 mm in (A,B), 50 mm in (C,D).
S. Jander, G. Stoll / Journal of Neuroimmunology 114 (2001) 253 – 258
3.3. Serum and CSF levels of IL-18 in GBS patients and NIND controls IL-18 levels in the serum of NIND patients [4267 pg / ml (mean6S.E.M.), range: 7–84 pg / ml] matched those given by the manufacturer for a collection of 60 control sera (mean 43 pg / ml, range: ,15–86 pg / ml). In GBS patients, IL-18 serum levels (Fig. 3A) were significantly
257
increased (238671 pg / ml, P,0.001). Similarly, CSF levels of IL-18 (Fig. 3B) were significantly higher in GBS patients than in NIND controls (3265 vs. 1962 pg / ml, P,0.001). However, IL-18 index values calculated as a measure of intrathecally released IL-18 (Fig. 3C) were significantly lower in GBS patients (0.0360.01) than in NIND controls (0.1160.02, P,0.001).
4. Discussion
Fig. 3. Box and whiskers plots illustrating absolute serum (A) and CSF (B) levels (pg / ml) as well as IL-18 index values (C, as a measure of intrathecally released IL-18) in NIND and GBS patients.
Acute immune-mediated demyelination in the PNS has been suggested to be due to a dysregulation of the cytokine network with a predominance of proinflammatory Th1mediated effector pathways (Hartung et al., 1990; Zhu et al., 1998). IL-18 is a potent Th1-inducing cytokine (Okamura et al., 1995) that has been implicated in the pathogenesis of Th1-mediated CNS diseases such as experimental autoimmune encephalomyelitis (EAE) (Wildbaum et al., 1998). In our present study we found an induction of IL-18 mRNA and protein in the nerve roots of EAN rats that peaked during active disease progression and paralleled the time course of T cell infiltration (Schmidt et al., 1992) and IFN-g expression (Gillen et al., 1998) in this model. In line with studies suggesting macrophages / monocytes as major cellular sources of IL-18 (Puren et al., 1999) IL-18 protein in EAN roots was mainly localized to ED11 macrophages. Taken together, our data therefore suggest a role for macrophage-derived IL-18 in the pathogenesis of Th1-mediated autoimmune demyelination in the PNS. As an extension of our results in the EAN model we found significantly elevated IL-18 serum levels in GBS patients. In line with previous reports showing elevation of the proinflammatory cytokines TNF-a (Creange et al., 1996) and IL-2 (Hartung et al., 1991) our finding further supports the notion of a strong systemic immune activation in GBS. Overall, there was considerable variation of IL-18 serum levels. The highest IL-18 level (1232 pg / ml) was found in a patient with severe disease requiring mechanical ventilation. However, attempts to systematically correlate IL-18 levels to clinical features such as duration and severity of disease at the time of presentation did not yield significant results in our relatively small sample. With respect to the elevated CSF concentrations of IL-18 in GBS, calculation of IL-18 index values as a measure of intrathecal cytokine production revealed a significant decrease in GBS relative to NIND controls. Thus, most IL-18 present in the CSF of GBS patients had most likely diffused passively from the circulation via the compromised blood / CSF barrier. Cleavage by caspase-1 has been shown to be critically important for the conversion of the inactive IL-18 precursor molecule into its mature active form (Ghayur et al., 1997; Gu et al., 1997). In the EAN model we found that
258
S. Jander, G. Stoll / Journal of Neuroimmunology 114 (2001) 253 – 258
caspase-1 mRNA increased in parallel with the levels of IL-18 mRNA. Although these studies were restricted to mRNA analysis and the ELISA assay used in our study did not differentiate between inactive and active cytokine protein it is likely that the coordinated induction of caspase-1 and IL-18 leads to the production of bioactive IL-18 that in turn promotes Th1-mediated disease activity. This view is supported by studies in EAE showing reduced disease incidence as well as defective maturation of CNS autoantigen-specific Th1 cells after administration of caspase-1 inhibitors (Furlan et al., 1999a). Preliminary findings in multiple sclerosis moreover indicate that the levels of caspase-1 mRNA in peripheral blood correlate with disease activity (Furlan et al., 1999b). Thus, interference with caspase-1 activity may provide a novel therapeutic strategy for downregulating proinflammatory cytokine activity in the treatment of autoimmune CNS and PNS diseases.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (Sto 162 / 8-1). G. Stoll holds a Hermannand Lilly-Schilling professorship. We thank Birgit Blomenkamp and Annette Tries for excellent technical assistance.
References Abbas, A.K., Murphy, K.M., Sher, A., 1996. Functional diversity of helper T lymphocytes. Nature 383, 787–793. Bazan, J.F., Timans, J.C., Kastelein, R.A., 1996. A newly defined interleukin-1? Nature 379, 591. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. Creange, A., Belec, L., Clair, B., Raphael, J.C., Gherardi, R.K., 1996. Circulating tumor necrosis factor (TNF)-alpha and soluble TNF-alpha receptors in patients with Guillain–Barre´ syndrome. J. Neuroimmunol. 68, 95–99. Furlan, R., Filippi, M., Bergami, A., Rocca, M.A., Martinelli, V., Poliani, P.L., Grimaldi, L.M., Desina, G., Comi, G., Martino, G., 1999b. Peripheral levels of caspase-1 mRNA correlate with disease activity in patients with multiple sclerosis; a preliminary study. J. Neurol. Neurosurg. Psychiatry 67, 785–788. Furlan, R., Martino, G., Galbiati, F., Poliani, P.L., Smiroldo, S., Bergami, A., Desina, G., Comi, G., Flavell, R., Su, M.S., Adorini, L., 1999a. Caspase-1 regulates the inflammatory process leading to autoimmune demyelination. J. Immunol. 163, 2403–2409. Ghayur, T., Banerjee, S., Hugunin, M., Butler, D., Herzog, L., Carter, A., Quintal, L., Sekut, L., Talanian, R., Paskind, M., Wong, W., Kamen, R., Tracey, D., Allen, H., 1997. Caspase-1 processes IFN-gamma-
inducing factor and regulates LPS-induced IFN-gamma production. Nature 386, 619–623. Gillen, C., Jander, S., Stoll, G., 1998. The sequential expression of mRNA for proinflammatory cytokines and interleukin-10 in the rat peripheral nervous system: comparison between in immune-mediated demyelination and Wallerian degeneration. J. Neurosci. Res. 51, 489–496. Gu, Y., Kuida, K., Tsutsui, H., Ku, G., Hsiao, K., Fleming, M.A., Hayashi, N., Higashino, K., Okamura, H., Nakanishi, K., Kurimoto, M., Tanimoto, T., Flavell, R.A., Sato, V., Harding, M.W., Livingston, D.J., Su, M.S.S., 1997. Activation of interferon-gamma inducing factor mediated by interleukin-1b converting enzyme. Science 275, 206–209. Hartung, H.P., Reiners, K., Schmidt, B., Stoll, G., Toyka, K.V., 1991. Serum interleukin-2 concentrations in Guillain–Barre´ syndrome and chronic idiopathic demyelinating polyradiculoneuropathy: comparison with other neurological diseases of presumed immunopathogenesis. Ann. Neurol. 30, 48–53. ¨ Hartung, H.P., Schafer, B., Van der Meide, P.H., Fierz, W., Heininger, K., Toyka, K.V., 1990. The role of interferon-gamma in the pathogenesis of experimental autoimmune disease of the peripheral nervous system. Ann. Neurol. 27, 247–257. Hartung, H.P., Toyka, K.V., 1990. T-cell and macrophage activation in experimental autoimmune neuritis and Guillain–Barre´ syndrome. Ann. Neurol. 27 (Suppl.), S57–S63. Hughes, R.A., Hadden, R.D., Gregson, N.A., Smith, K.J., 1999. Pathogenesis of Guillain–Barre´ syndrome. J. Neuroimmunol. 100, 74–97. Jander, S., Stoll, G., 1998. Differential induction of interleukin-12, interleukin-18, and interleukin-1beta converting enzyme mRNA in experimental autoimmune encephalomyelitis of the Lewis rat. J. Neuroimmunol. 91, 93–99. Kohno, K., Kurimoto, M., 1998. Interleukin 18, a cytokine which resembles IL-1 structurally and IL-12 functionally but exerts its effect independently of both. Clin. Immunol. Immunopathol. 86, 11–15. Okamura, H., Tsutsui, H., Komatsu, T., Yutsudo, M., Hakura, A., Tanimoto, T., Torigoe, K., Okura, T., Nukada, Y., Hattori, K., Akita, K., Namba, M., Tanabe, F., Konishi, K., Fukuda, S., Kurimoto, M., 1995. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature 378, 88–91. Puren, A.J., Fantuzzi, G., Dinarello, C.A., 1999. Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells. Proc. Natl. Acad. Sci. USA 96, 2256–2261. Schmidt, B., Stoll, G., Van der Meide, P.H., Jung, S., Hartung, H.P., 1992. Transient cellular expression of gamma-interferon in myelin-induced and T-cell line mediated experimental autoimmune neuritis. Brain 115, 1633–1646. Stoll, G., Hartung, H.P., 1992. The role of macrophages in degeneration and immune mediated demyelination of the peripheral nervous system. Adv. Neuroimmunol. 2, 163–179. Trinchieri, G., 1993. Interleukin-12 and its role in the generation of Th1 cells. Immunol. Today 14, 335–338. Wildbaum, G., Youssef, S., Grabie, N., Karin, N., 1998. Neutralizing antibodies to IFN-gamma-inducing factor prevent experimental autoimmune encephalomyelitis. J. Immunol. 161, 6368–6374. Zhu, J., Mix, E., Link, H., 1998. Cytokine production and the pathogenesis of experimental autoimmune neuritis and Guillain–Barre´ syndrome. J. Neuroimmunol. 84, 40–52.
Interleukin-18 is induced in acute inflammatory demyelinating polyneuropathy Sebastian Jander*, Guido Stoll ¨ , Germany Department of Neurology, Heinrich-Heine-University, Moorenstraße 5, D-40225 Dusseldorf Received 20 September 2000; received in revised form 5 December 2000; accepted 12 December 2000
Abstract T lymphocytes of the Th1 subset producing the proinflammatory cytokine interferon-g (IFN-g) have been implicated in the pathogenesis of immune-mediated diseases of the peripheral nervous system (PNS) such as the acute Guillain–Barre´ syndrome (GBS) and its animal model experimental autoimmune neuritis (EAN). Interleukin-18 (IL-18) is a potent IFN-g-inducing cytokine that is synthesized as an inactive precursor molecule and cleaved by caspase-1 into its mature active form. In our present study we analyzed the expression of IL-18 and caspase-1 in the nerve roots of EAN rats using reverse transcriptase–polymerase chain reaction and immunocytochemistry. Using an enzyme-linked immunosorbent assay, we furthermore determined IL-18 protein levels in paired serum and cerebrospinal fluid (CSF) samples from patients with GBS as well as from noninflammatory neurologic disease (NIND) controls. In EAN, IL-18 and caspase-1 mRNA levels in the nerve roots increased during the stage of active disease progression. Immunocytochemically, both perivascular and parenchymal IL-18 protein expression was increased in the roots of EAN rats and mainly associated with ED11 macrophages stained on serial sections. IL-18 serum levels were significantly higher in GBS patients than in NIND controls (238671 vs. 4267 pg / ml, P,0.001). Our data implicate the Th1-inducing cytokine IL-18 in the pathogenesis of acute immune-mediated PNS demyelination. 2001 Elsevier Science B.V. All rights reserved. Keywords: Peripheral nervous system; Cytokine; Demyelination; Interleukin-18; Interferon-g; Caspase-1; Th1 cells
1. Introduction The Guillain–Barre´ syndrome (GBS) is an acute inflammatory demyelinating polyneuropathy that frequently occurs as a sequela of gastrointestinal infections and leads to progressive ascending tetraparesis (Hughes et al., 1999). Several aspects of the disease are mimicked in the model of experimental autoimmune neuritis (EAN) which can be induced in susceptible rat strains by sensitization against peripheral nervous system (PNS) myelin constituents such as the P2 protein (Hartung and Toyka, 1990). Pathophysiological studies have suggested that T helper cell-dependent activation of macrophages is the principal mechanism underlying immune-mediated myelin destruction in GBS and EAN (Hartung and Toyka, 1990; Stoll and Hartung, 1992). Depending on their specific pattern of cytokine production T helper cells can be subdivided into
*Corresponding author. Tel.: 149-211-81-18978; fax: 149-211-8118485. E-mail address: [email protected] (S. Jander).
two major subpopulations, i.e. Th1 cells secreting the proinflammatory cytokines interferon (IFN)-g and tumor necrosis factor-a and Th2 cells secreting antiinflammatory cytokines such as interleukin (IL)-4 and IL-10 (Abbas et al., 1996). In EAN, T cells infiltrating nerve roots abundantly express IFN-g mRNA and protein (Gillen et al., 1998; Schmidt et al., 1992). Therefore, it is a widely accepted view that disease induction in the EAN model as well as in human GBS follows a Th1-dependent immunopathogenesis (Zhu et al., 1998). The generation and activation of Th1 cells depends on the appropriate delivery of specific costimulatory signals during initial stages of a cellular immune response (Abbas et al., 1996). Antigen-presenting cells such as activated macrophages produce the immunoregulatory cytokines IL12 and IL-18 which are potent IFN-g-inducing factors and thereby favor a Th1-like polarization of T cell-mediated immune responses (Trinchieri, 1993; Kohno and Kurimoto, 1998). IL-18 was discovered only recently in a mouse model of endotoxic liver injury (Okamura et al., 1995). IL-18 is synthesized as an inactive precursor molecule that exhibits structural homology to IL-1b
0165-5728 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0165-5728( 00 )00460-4
254
S. Jander, G. Stoll / Journal of Neuroimmunology 114 (2001) 253 – 258
(Bazan et al., 1996) and accordingly has to be cleaved by caspase-1 (previously termed IL-1b converting enzyme) into its mature active form (Ghayur et al., 1997; Gu et al., 1997). In experimental autoimmune encephalomyelitis (EAE) a coordinated increase of both IL-18 and caspase-1 mRNA occurs during active disease progression (Jander and Stoll, 1998) suggesting a crucial role of this cytokine pathway in disease induction. So far, the mechanisms initiating the Th1-mediated immune response in acute inflammatory polyneuropathies are largely unknown. In our present study we have used reverse transcriptase–polymerase chain reaction (RT– PCR) and immunocytochemistry to analyze the expression of IL-18 and caspase-1 in nerve roots during the course of actively induced EAN in Lewis rats. To further substantiate a possible role of IL-18 in the pathogenesis of human PNS disease we have additionally measured serum and cerebrospinal fluid (CSF) levels of IL-18 in patients with GBS.
2. Methods
2.1. Animal experiments EAN was induced in 8-week-old female Lewis rats by immunization with bovine PNS myelin as described previously (Gillen et al., 1998). Disease severity was scored as follows: 0, no clinical signs; 0.5, loss of tail tone; 1, complete tail paresis; 2, incomplete hind limb paresis; 3, complete paraplegia. For RT–PCR analysis, EAN animals along with normal controls (n53–4 in each group) were sacrificed on days 11, 13, 15, 21, and 28 by an overdose of ether. Nerve roots were prepared rapidly, snap frozen in liquid nitrogen, and stored at 2808C until RNA preparation. For immunocytochemistry, additional rats (n53 in each group) were perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The lumbosacral spinal cord with adhering nerve roots was prepared, postfixed overnight in the same fixative, and embedded into paraffin.
2.2. RT–PCR Total RNA was isolated from the nerve roots according to standard procedures (Chomczynski and Sacchi, 1987) and reverse transcribed into cDNA as detailed elsewhere (Gillen et al., 1998). cDNA equivalent to 20 ng of total RNA was subjected to subsequent PCR analysis using primers specific for IL-18, caspase-1, or the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as described previously (Jander and Stoll, 1998). In preliminary experiments, optimal cycling con-
ditions were established allowing amplification of each cDNA in the linear range. Cycle numbers were as follows: IL-18528; caspase-1529; and GAPDH520. PCR products were separated on 1.5% agarose gels containing 10 mg / ml ethidium bromide, photographed using a CSCcamera (Cybertech, Berlin, Germany) and densitometric analysis was performed with TINA 2.1 software (Raytest, Straubenhardt, Germany).
2.3. Immunocytochemistry Longitudinal 5 mm sections of paraffin-embedded nerve roots were deparaffinized in xylene and rehydrated in a descending series of ethanol. After blocking of nonspecific binding sites with 3% normal horse serum, goat anti-rat IL-18 primary antibody (R&D Systems, Minneapolis, MN) at 2 mg / ml final working concentration was incubated overnight at 48C. Detection was performed with biotinylated horse anti-goat secondary antibody (Vector Laboratories, Burlingame, CA) and the ABC Elite kit (Vector) with diaminobenzidine as substrate. For cellular identification, adjacent serial sections were incubated with mAb ED1 against phagocytic macrophages (Serotec, Oxford, UK) at 1:2000 dilution, followed by biotinylated horse anti-mouse secondary antibody (Vector) and detection reagents as above. As control, replacement of primary antibodies with irrelevant goat or mouse IgG led to a disappearance of immunostaining.
2.4. Patients A total of 36 individuals were studied. Paired serum and CSF samples were collected between 1995 and 1999 and stored at 2708C. Before freezing, CSF samples were centrifuged for 5 min at high speed to remove cells. Eighteen patients met the established diagnostic criteria for acute GBS. All had rapidly progressive motor weakness of lower and upper extremities, areflexia, and mild sensory signs. Routine CSF studies showed normal cell counts in all patients. CSF protein content was elevated in 13, but normal in five subjects. Electrophysiologic measurements suggested generalized or focal demyelination in 15 cases, while predominantly axonal affection was found in three patients. As a control group [noninflammatory neurologic diseases (NIND)] 18 patients with one of the following diagnoses were studied: tension headache, depression, normal pressure hydrocephalus, migraine, disc herniation, pseudotumor cerebri, ischemic stroke, epilepsy, subcortical arteriosclerotic encephalopathy, Alzheimer’s disease. In all NIND patients, routine CSF analysis was normal and review of the clinical data revealed no evidence for systemic or localized inflammatory, infectious, or malignant disease.
S. Jander, G. Stoll / Journal of Neuroimmunology 114 (2001) 253 – 258
2.5. Enzyme-linked immunosorbent assay ( ELISA)
3. Results
The QuantikineE human IL-18 ELISA kit (R&D Systems, Minneapolis, MN) was used according to the manufacturer’s instructions. Both serum and CSF samples were tested at 1:2 dilution. To correct CSF IL-18 levels for blood CSF-barrier dysfunction typical in GBS we calculated IL-18 index values as follows: [IL-18 CSF / IL-18 serum :albumin CSF / albumin serum ].
3.1. Expression of IL-18 and caspase-1 mRNA during EAN
For statistical analysis, Mann–Whitney U-test was performed using GraphPad Prism 3.00 software (GraphPad Software, San Diego, CA). P-values,0.05 were considered as statistically significant.
255
Following immunization with bovine PNS myelin the animals developed tail paralysis as the first sign of EAN at days 10–11 after immunization, progressed until day 15 when complete paraparesis of the hind limbs was reached, and recovered spontaneously thereafter (Fig. 1A). On day 28, all animals were asymptomatic. In the nerve roots of normal control rats low constitutive levels of both IL-18 and caspase-1 mRNA were detected. Both mRNAs increased above baseline levels at the onset
Fig. 1. (A) Clinical course of actively induced EAN in Lewis rats. (B,C) RT–PCR analysis of IL-18 and caspase-1 mRNA levels in nerve roots during the course of actively induced EAN in Lewis rats. (B) Representative original RT–PCR findings. GAPDH was used as ‘house-keeping gene’ control in subsequent semiquantitative analysis. (C) Levels of IL-18 and caspase-1 mRNA relative to those of the ‘house-keeping gene’ GAPDH in n53–4 animals on each time point. Bars denote mean6S.E.M.
256
S. Jander, G. Stoll / Journal of Neuroimmunology 114 (2001) 253 – 258
of clinical disease at day 11 after immunization (Fig. 1B,C). IL-18 mRNA levels continued to increase until day 13 and showed a protracted decrease thereafter. Levels of caspase-1 mRNA decreased somewhat more rapidly already at day 13 after immunization. However, both messages remained elevated above baseline levels until day 28.
3.2. Immunocytochemical localization of IL-18 protein in inflamed nerve roots To corroborate our mRNA findings at the protein level we performed immunocytochemical experiments using an affinity-purified polyclonal antibody against rat IL-18. In
control nerves, weak constitutive expression on longitudinally orientated cells resembling resident PNS macrophages was found. In line with the PCR data a clear increase of IL-18 immunoreactivity occurred at days 11 and 13 after immunization with a subsequent decline beyond day 15 (Fig. 2A–D). At high magnification, IL-18 induction appeared to be mainly associated with perivascular infiltrates (Fig. 2B), but was also observed on parenchymal cells (Fig. 2D). At both sites, staining of serial sections revealed extensive colocalization of IL-18 immunoreactivity with the phagocyte marker ED1 (Fig. 2A,C) suggesting that IL-18 in EAN roots was mainly expressed by activated macrophages.
Fig. 2. Immunocytochemical localization of IL-18 protein on longitudinal paraffin sections of EAN roots on day 11 after immunization. A,B as well as C,D are taken from pairs of adjacent serial sections stained for the macrophage marker ED1 (A,C) and IL-18 (B,D), respectively. Extensive colocalization between both antigens is evident in perivascular infiltrates (A,B) as well as parenchymal cells (C,D). Arrowheads in C and D point to cells that coexpress IL-18 and ED1 antigen. Bars: 25 mm in (A,B), 50 mm in (C,D).
S. Jander, G. Stoll / Journal of Neuroimmunology 114 (2001) 253 – 258
3.3. Serum and CSF levels of IL-18 in GBS patients and NIND controls IL-18 levels in the serum of NIND patients [4267 pg / ml (mean6S.E.M.), range: 7–84 pg / ml] matched those given by the manufacturer for a collection of 60 control sera (mean 43 pg / ml, range: ,15–86 pg / ml). In GBS patients, IL-18 serum levels (Fig. 3A) were significantly
257
increased (238671 pg / ml, P,0.001). Similarly, CSF levels of IL-18 (Fig. 3B) were significantly higher in GBS patients than in NIND controls (3265 vs. 1962 pg / ml, P,0.001). However, IL-18 index values calculated as a measure of intrathecally released IL-18 (Fig. 3C) were significantly lower in GBS patients (0.0360.01) than in NIND controls (0.1160.02, P,0.001).
4. Discussion
Fig. 3. Box and whiskers plots illustrating absolute serum (A) and CSF (B) levels (pg / ml) as well as IL-18 index values (C, as a measure of intrathecally released IL-18) in NIND and GBS patients.
Acute immune-mediated demyelination in the PNS has been suggested to be due to a dysregulation of the cytokine network with a predominance of proinflammatory Th1mediated effector pathways (Hartung et al., 1990; Zhu et al., 1998). IL-18 is a potent Th1-inducing cytokine (Okamura et al., 1995) that has been implicated in the pathogenesis of Th1-mediated CNS diseases such as experimental autoimmune encephalomyelitis (EAE) (Wildbaum et al., 1998). In our present study we found an induction of IL-18 mRNA and protein in the nerve roots of EAN rats that peaked during active disease progression and paralleled the time course of T cell infiltration (Schmidt et al., 1992) and IFN-g expression (Gillen et al., 1998) in this model. In line with studies suggesting macrophages / monocytes as major cellular sources of IL-18 (Puren et al., 1999) IL-18 protein in EAN roots was mainly localized to ED11 macrophages. Taken together, our data therefore suggest a role for macrophage-derived IL-18 in the pathogenesis of Th1-mediated autoimmune demyelination in the PNS. As an extension of our results in the EAN model we found significantly elevated IL-18 serum levels in GBS patients. In line with previous reports showing elevation of the proinflammatory cytokines TNF-a (Creange et al., 1996) and IL-2 (Hartung et al., 1991) our finding further supports the notion of a strong systemic immune activation in GBS. Overall, there was considerable variation of IL-18 serum levels. The highest IL-18 level (1232 pg / ml) was found in a patient with severe disease requiring mechanical ventilation. However, attempts to systematically correlate IL-18 levels to clinical features such as duration and severity of disease at the time of presentation did not yield significant results in our relatively small sample. With respect to the elevated CSF concentrations of IL-18 in GBS, calculation of IL-18 index values as a measure of intrathecal cytokine production revealed a significant decrease in GBS relative to NIND controls. Thus, most IL-18 present in the CSF of GBS patients had most likely diffused passively from the circulation via the compromised blood / CSF barrier. Cleavage by caspase-1 has been shown to be critically important for the conversion of the inactive IL-18 precursor molecule into its mature active form (Ghayur et al., 1997; Gu et al., 1997). In the EAN model we found that
258
S. Jander, G. Stoll / Journal of Neuroimmunology 114 (2001) 253 – 258
caspase-1 mRNA increased in parallel with the levels of IL-18 mRNA. Although these studies were restricted to mRNA analysis and the ELISA assay used in our study did not differentiate between inactive and active cytokine protein it is likely that the coordinated induction of caspase-1 and IL-18 leads to the production of bioactive IL-18 that in turn promotes Th1-mediated disease activity. This view is supported by studies in EAE showing reduced disease incidence as well as defective maturation of CNS autoantigen-specific Th1 cells after administration of caspase-1 inhibitors (Furlan et al., 1999a). Preliminary findings in multiple sclerosis moreover indicate that the levels of caspase-1 mRNA in peripheral blood correlate with disease activity (Furlan et al., 1999b). Thus, interference with caspase-1 activity may provide a novel therapeutic strategy for downregulating proinflammatory cytokine activity in the treatment of autoimmune CNS and PNS diseases.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (Sto 162 / 8-1). G. Stoll holds a Hermannand Lilly-Schilling professorship. We thank Birgit Blomenkamp and Annette Tries for excellent technical assistance.
References Abbas, A.K., Murphy, K.M., Sher, A., 1996. Functional diversity of helper T lymphocytes. Nature 383, 787–793. Bazan, J.F., Timans, J.C., Kastelein, R.A., 1996. A newly defined interleukin-1? Nature 379, 591. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. Creange, A., Belec, L., Clair, B., Raphael, J.C., Gherardi, R.K., 1996. Circulating tumor necrosis factor (TNF)-alpha and soluble TNF-alpha receptors in patients with Guillain–Barre´ syndrome. J. Neuroimmunol. 68, 95–99. Furlan, R., Filippi, M., Bergami, A., Rocca, M.A., Martinelli, V., Poliani, P.L., Grimaldi, L.M., Desina, G., Comi, G., Martino, G., 1999b. Peripheral levels of caspase-1 mRNA correlate with disease activity in patients with multiple sclerosis; a preliminary study. J. Neurol. Neurosurg. Psychiatry 67, 785–788. Furlan, R., Martino, G., Galbiati, F., Poliani, P.L., Smiroldo, S., Bergami, A., Desina, G., Comi, G., Flavell, R., Su, M.S., Adorini, L., 1999a. Caspase-1 regulates the inflammatory process leading to autoimmune demyelination. J. Immunol. 163, 2403–2409. Ghayur, T., Banerjee, S., Hugunin, M., Butler, D., Herzog, L., Carter, A., Quintal, L., Sekut, L., Talanian, R., Paskind, M., Wong, W., Kamen, R., Tracey, D., Allen, H., 1997. Caspase-1 processes IFN-gamma-
inducing factor and regulates LPS-induced IFN-gamma production. Nature 386, 619–623. Gillen, C., Jander, S., Stoll, G., 1998. The sequential expression of mRNA for proinflammatory cytokines and interleukin-10 in the rat peripheral nervous system: comparison between in immune-mediated demyelination and Wallerian degeneration. J. Neurosci. Res. 51, 489–496. Gu, Y., Kuida, K., Tsutsui, H., Ku, G., Hsiao, K., Fleming, M.A., Hayashi, N., Higashino, K., Okamura, H., Nakanishi, K., Kurimoto, M., Tanimoto, T., Flavell, R.A., Sato, V., Harding, M.W., Livingston, D.J., Su, M.S.S., 1997. Activation of interferon-gamma inducing factor mediated by interleukin-1b converting enzyme. Science 275, 206–209. Hartung, H.P., Reiners, K., Schmidt, B., Stoll, G., Toyka, K.V., 1991. Serum interleukin-2 concentrations in Guillain–Barre´ syndrome and chronic idiopathic demyelinating polyradiculoneuropathy: comparison with other neurological diseases of presumed immunopathogenesis. Ann. Neurol. 30, 48–53. ¨ Hartung, H.P., Schafer, B., Van der Meide, P.H., Fierz, W., Heininger, K., Toyka, K.V., 1990. The role of interferon-gamma in the pathogenesis of experimental autoimmune disease of the peripheral nervous system. Ann. Neurol. 27, 247–257. Hartung, H.P., Toyka, K.V., 1990. T-cell and macrophage activation in experimental autoimmune neuritis and Guillain–Barre´ syndrome. Ann. Neurol. 27 (Suppl.), S57–S63. Hughes, R.A., Hadden, R.D., Gregson, N.A., Smith, K.J., 1999. Pathogenesis of Guillain–Barre´ syndrome. J. Neuroimmunol. 100, 74–97. Jander, S., Stoll, G., 1998. Differential induction of interleukin-12, interleukin-18, and interleukin-1beta converting enzyme mRNA in experimental autoimmune encephalomyelitis of the Lewis rat. J. Neuroimmunol. 91, 93–99. Kohno, K., Kurimoto, M., 1998. Interleukin 18, a cytokine which resembles IL-1 structurally and IL-12 functionally but exerts its effect independently of both. Clin. Immunol. Immunopathol. 86, 11–15. Okamura, H., Tsutsui, H., Komatsu, T., Yutsudo, M., Hakura, A., Tanimoto, T., Torigoe, K., Okura, T., Nukada, Y., Hattori, K., Akita, K., Namba, M., Tanabe, F., Konishi, K., Fukuda, S., Kurimoto, M., 1995. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature 378, 88–91. Puren, A.J., Fantuzzi, G., Dinarello, C.A., 1999. Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells. Proc. Natl. Acad. Sci. USA 96, 2256–2261. Schmidt, B., Stoll, G., Van der Meide, P.H., Jung, S., Hartung, H.P., 1992. Transient cellular expression of gamma-interferon in myelin-induced and T-cell line mediated experimental autoimmune neuritis. Brain 115, 1633–1646. Stoll, G., Hartung, H.P., 1992. The role of macrophages in degeneration and immune mediated demyelination of the peripheral nervous system. Adv. Neuroimmunol. 2, 163–179. Trinchieri, G., 1993. Interleukin-12 and its role in the generation of Th1 cells. Immunol. Today 14, 335–338. Wildbaum, G., Youssef, S., Grabie, N., Karin, N., 1998. Neutralizing antibodies to IFN-gamma-inducing factor prevent experimental autoimmune encephalomyelitis. J. Immunol. 161, 6368–6374. Zhu, J., Mix, E., Link, H., 1998. Cytokine production and the pathogenesis of experimental autoimmune neuritis and Guillain–Barre´ syndrome. J. Neuroimmunol. 84, 40–52.