Acute effects of interleukin-1β on noradrenaline release from the human neuroblastoma cell line SH-SY5Y

Neuroscience Letters 251 (1998) 89–92

Acute effects of interleukin-1b on noradrenaline release from the human neuroblastoma cell line SH-SY5Y Elizabeth L.K. Smith a, Atticus H. Hainsworth a , b ,* a

School of Life Sciences, University of Greenwich, London SE18 6PF, UK b School of Pharmacy, DeMontfort University, Leicester, LE1 9BH, UK

Received 23 June 1997; received in revised form 3 June 1998; accepted 8 June 1998

Abstract Interleukins are potent intercellular messenger peptides, initially found in cells of the immune system and best known for producing chronic, genomic effects in target cells. Here, interleukin-1b (IL-1b) was tested for acute effects on neurotransmitter release. The human neuroblastoma-derived cell-line SH-SY5Y is a model for mature post-ganglionic sympathetic neurones and release of tritiated noradrenaline from these cells was measured, in response to stimulation with either elevated extracellular K+ concentration (100 K+) or veratridine. Pre-incubation for 15–25 min with 60 pM (but not 0.06 pM) IL-1b significantly reduced 100 K+-evoked release (by approximately 75%). The interleukin was without effect on basal or veratridine-evoked noradrenaline release. The present data suggest two distinct stimulatory pathways: one that is activated by 100 K+ and veratridine and is unaffected by IL-1b and another that is activated by 100 K+ but not veratridine and is inhibited by IL-1b. The acute depression of 100 K+-evoked transmitter release may be involved in immune system–nervous system interactions.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Interleukin-1b; Catecholamines; Norepinephrine; Calcium channels; Inflammatory cytokines

Interactions between the immune system and the nervous system, principally through autonomic or neuroendocrine pathways, are increasingly apparent [1,10,17]. Some of these interactions are likely to be mediated by the interleukins, potent peptide messengers initially found in cells of the immune system and best known for their chronic effects on target cells. The 17-kDa interleukin 1b (IL-1b) is a classic inflammatory cytokine, released by peripheral blood monocytes and macrophages following inflammation or cellular stress [5,6]. It is also found in the CNS and in sympathetic neurones [14], levels of IL-1b mRNA in the CNS being elevated within 15 min of central ischaemia [12]. IL-1b is implicated in numerous effects on the nervous system, including fever induction (via release of corticotrophin-releasing hormone) and hyperalgesia to inflammatory and sympathetic pain [12,14] and previous work has indi-

* Corresponding author. Tel.: +44 116 2551551; fax: +44 116 2577287.

cated modulation of neurotransmitter release by IL-1b [4,9, 11]. The present study tested for acute effects of IL-1b in a well-defined model of neurotransmitter release, the human neuroblastoma cell-line SH-SY5Y. These cells exhibit many features of mature post-ganglionic sympathetic neurones, including neuronal morphology, calcium-dependent noradrenaline release and functional receptors for endogenous ligands, including acetylcholine, bradykinin and neuropeptide Y [8,19]. SH-SY5Y cells (ECACC, Porton Down, UK) were cultured in a 1:1 mixture of F-12 Ham’s medium and Eagle’s minimal essential medium with Earle’s salts, supplemented with L-glutamine and 10% foetal calf serum. Cells (passages 11–19) were grown to confluence in 12- or 24-well plates at a high density, in a 95% air/5% CO2 humidified incubator at 37°C and used in experiments 4–7 days after plating. All cell culture solutions came from GIBCO Life Technologies, Glasgow, UK. HEPES-buffered-saline (HBS) was made up freshly each day and contained (mM): 140 NaCl,

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00474- 1

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2.5 KCl, 10 HEPES acid, 2.5 CaCl2, 0.5 MgSO4, 1 NaH2 PO4, and 10 glucose (pH 7.4) with 1 M NaOH. High-potassium HBS (100 K+) was made up identically to HBS but with 40 mM NaCl and 100 mM KCl. [3H]noradrenaline (supplied in 0.02 M acetic acid: ethanol 9:1, 37 MBq/ml, Amersham, UK) was stored at 4°C and diluted 500× into HBS just before use. All solutions to which cells were exposed were kept at 37°C and were supplemented with 0.2 mM ascorbate (to reduce oxidation of noradrenaline) and 0.2 mM pargyline (to block the activity of monoamine oxidase, which deaminates noradrenaline). Aqueous stock solutions of human IL-1b (10 mg/ml; R and D Systems, Abingdon UK) and veratridine (10 mM) were stored at −20°C and a 2 mM ethanolic stock of A 23187 was stored at 4°C; each was diluted to the working concentration in HBS just before use. All drugs were from Sigma, Poole, UK unless otherwise stated. This release assay is based on that of Murphy et al. [8]. In each experiment, cell layers were gently rinsed twice with HBS (usually 1 ml/well), then incubated with HBS containing tritiated noradrenaline (74 kBq per well) for 60–90 min to load the cells with labelled neurotransmitter. After this loading period, cells were rinsed seven times with HBS for 3, 60, 5, 5, 5, 5 and 5 min periods respectively, to remove the remaining extracellular [3H]noradrenaline, then exposed to HBS containing IL-1b, either 0 or 100 pg/ml or 10 ng/ml for 15–25 min. The following excitatory stimuli were then applied to different wells: (1) 100 K+, (2) 50 mM veratridine,

(3) no stimulus (control HBS), each containing either 0 or 100 pg/ml or 10 ng/ml IL-1b. These stimuli were applied for 7–15 min in different experiments, and the well contents collected in numbered scintillation vials. Finally, all remaining [3H]noradrenaline was extracted from the cells with 0.1 M perchloric acid for 20 min and this acid-extract collected in numbered vials. The 3H-containing sample in each vial was shaken with a 1:8 excess of Ecoscint A scintillant fluid (National Diagnostics, Hessle, UK), darkadapted for 60 min, then assayed for beta emission using a liquid scintillation analyser (Canberra Packard Instrument TRI-CARB 2000CA). The vials were counted as soon as possible after the samples were collected, to avoid loss in efficacy by photon, chemical or colour quenching. Each vial was counted for 3 min with automatic luminescence correction. The measured [3H]noradrenaline release was determined as a percentage of the total [3H]noradrenaline present at the start of a sample period. Mean and SD of release was computed over triplicate wells and mean basal percentage release subtracted from all samples within a given experiment. Pooled mean data and SEM of at least three experiments were calculated (see Fig. 1) and significance (P , 0.05) tested using paired, two-tailed t-tests. In the absence of excitatory stimuli, [3H]noradrenaline release was not significantly affected by acute exposure (15–25 min) to IL-1b (not shown). After subtracting basal release in the absence of interleukin, the calculated mean

Fig. 1. Acute effect of interleukin-1b (IL-1b) on noradrenaline release from SH-SY5Y cells. Cell monolayers were loaded with tritiated noradrenaline, then stimulated with either elevated extracellular potassium (100 K+) or 50 mM veratridine (VERA) after 15–25 min pre-incubation with either 0, 0.06 or 60 pM IL-1b. Within each experiment, background-subtracted release was normalised with respect to interleukin-free conditions (control). Bars show mean and SEM of 3–6 experiments, each with triplicate wells (*P , 0.05).

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percent release in the presence of 100 pg/ml IL-1b (roughly equivalent to 0.06 pM) was −0.06 ± 0.74% (mean, SEM, n = 6) and in the presence of 10 ng/ml IL-1b (roughly equivalent to 60 pM) it was −1.4 ± 2.0% (n = 5). In the absence of interleukin, stimulation with elevated extracellular potassium (100 K+) produced 6.5 ± 3.4% release (n = 7) and that with veratridine (50 mM) 9.9 ± 1.2% (n = 3). The veratridine-evoked release is higher than in previous work [7,18] possibly due to the longer stimuli used here. Given this variability in response, results are shown in Fig. 1 normalised with respect to the average percent response in the absence of IL-1b. Potassium-induced release was negligibly affected by 0.06 pM IL-1b, but significantly reduced (by approximately 75%) by a relatively high concentration, 60 pM. Release in response to veratridine stimulation was not significantly affected by 0.06 pM or 60 pM IL-1b (Fig. 1). In two experiments the calcium ionophore A 23187 (1 mM) potently induced release that was significantly inhibited by 0.06 pM IL-1b, by approximately 30% (not shown). The ionophore allows entry of Ca2+ ions throughout the plasma membrane and SH-SY5Y cells are known to have vesicles at somatic sites [19], hence this inhibitory effect of IL-1b may reflect a non-exocytotic action, such as augmented cytoplasmic Ca2+ sequestration. The present results show a direct acute effect of the inflammatory cytokine IL-1b on neurotransmitter release, a fundamental neuronal function. Receptors for IL-1b are widespread and are known to be present in the cell-line SKN-SH, the precursor of SH-SY5Y [3]. Depolarisation with high extracellular K+, and removal of sodium-channel inactivation with veratridine, are standard stimuli for exocytotic release of noradrenaline from SH-SY5Y cells [2,7,8,18,19]. Both stimuli are thought to cause influx of calcium ions by opening voltage-dependent calcium channels (VDCCs), the former directly, the latter via action potential generation. Stimulus with 100 mM external K+ is likely to be an immediate and complete depolarisation, leading to rapid opening of all available VDCCs followed by inactivation. Veratridine probably provides a more gradual but maintained increase in action potential generation, and repeated VDCC activation at axon terminals. The present data suggest two distinct stimulatory pathways – possibly two subpopulations of VDCCs – that can lead to noradrenaline release: one that is activated by 100 K+ and veratridine and is unaffected by IL-1b and another that is activated by 100 K+ but not veratridine and is inhibited by IL-1b. Other workers find that 100 K+-evoked release is reduced by 76% by 5 mM nifedipine (assumed to be a selective antagonist for L-type VDCCs) and 47% by q-conotoxin GVIA (selective for N-type VDCCs) [7]. Thus it is possible that IL-1b selectively inhibits a secretory pathway dependent on L-type VDCCs. In rat ventricular myocytes, 30 pM IL-1b inhibited L-type VDCCs via the lipid messenger ceramide [15]. IL-1b also depressed calcium currents in guinea pig cardiac myocytes

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[13] and hippocampal neurones [11] and in molluscan neurones [16], inhibited long-term potentiation in rat hippocampus [4] and reduced K+ induced glutamate release from hippocampal synaptosomes [9]. By contrast, 50 pM IL-1b potentiated VDCC activity [20], in rat vascular smooth muscle cells. In conclusion, acute exposure to IL-1b inhibits potassium depolarisation evoked noradrenaline release from a sympathetic neurone-like cell-line. This phenomenon may be involved in interactions between the immune system and the autonomic nervous system. The authors are grateful to the UK Biotechnology and Biological Science Research Council for financial support (quota studentship to ELKS) and to Dr. P.F.T. Vaughan for helpful comments. [1] Ader, R., Cohen, N. and Felten, D., Psychoneuroimmunology: interactions between the nervous system and the immune system, Lancet, 345 (1995) 99–103. [2] Atcheson, R., Lambert, D.G., Hirst, R.A. and Rowbotham, D.J., Studies on the mechanism of [3H]-noradrenaline release from SH-SY5Y cells: the role of Ca and cyclic AMP, Br. J. Pharmacol., 111 (1994) 787–792. [3] Bossu, P., Ruggiero, P., Macchia, G., Maurizi, G., Bizzari, C., Neumann, D., Tagliabue, A. and Boraschi, D., Interaction between interleukin-1 and ciliary neurotrophic factor in the regulation of neuroblastoma cell functions, Eur. Cytokine Network, 8 (1997) 367–374. [4] Cunningham, A.J., Murray, C.A., O’Neill, L.A.J., Lynch, M.A. and O’Connor, J.J., Interleukin-1b (IL-1b) and tumour necrosis factor (TNF) inhibit long-term potentiation in the rat dentate gyrus in-vitro, Neurosci. Lett., 203 (1996) 17–20. [5] Dinarello, C.A., Biologic basis for interleukin-1 in disease, Blood, 87 (1996) 2095–2147. [6] Dinarello, C.A. and Wolff, S.M., The role of interleukin-1 in disease, N. Engl. J. Med., 328 (1993) 106–113. [7] McDonald, R.L., Vaughan, P.F.T. and Peers, C., Muscarinic (M1) receptor-mediated inhibition of K+-evoked [3H]-noradrenaline release from human neuroblastoma (SH-SY5Y) cells via inhibition of L- and N-type Ca2+ channels, Br. J. Pharmacol., 113 (1994) 621–627. [8] Murphy, N.P., Ball, S.G. and Vaughan, P.F.T., Potassium- and carbachol-evoked release of [3H]-noradrenaline from human neuroblastoma cells, SH-SY5Y, J. Neurochem., 56 (1991) 1811–1815. [9] Murray, C.A., McGahon, B., McBennett, S. and Lynch, M.A., Interleukin-1b inhibits glutamate release in hippocampus of young, but not aged, rats, Neurobiol. Aging, 18 (1997) 343– 348. [10] Pennisi, E., Neuroimmunology: tracing molecules that make the brain–body connection, Science, 275 (1997) 930–931. [11] Plata-Salaman, C.R. and ffrench-Mullen, J.M.H., Interleukin-1b inhibits Ca channel currents in hippocampal neurons through protein kinase C, Eur. J. Pharmacol., 266 (1994) 1–10. [12] Rothwell, N.J. and Hopkins, S.J., Cytokines and the nervous system: actions and mechanisms of action, Trends Pharm. Sci., 18 (1995) 130–136. [13] Rozanski, G.J. and Witt, R.C., IL-1 inhibits b-adrenergic control of cardiac calcium current: role of L-arginine/nitric oxide pathway, Am. J. Physiol., 267 (1994) H1753–H1758. [14] Schobitz, B., De Kloet, E.R. and Holsboer, F., Gene expression and function of interleukin-1, interleukin-6 and tumor necrosis factor in the brain, Prog. Neurobiol., 44 (1994) 397–432.

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