Selective Blockade of T Lymphocyte K Channels Ameliorates Experimental Autoimmune Encephalomyelitis, a Model for Multiple Sclero

Selective blockade of T lymphocyte K؉ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis Christine Beeton*†, Heike Wulff†‡, Jocelyne Barbaria*, Olivier Clot-Faybesse*, Michael Pennington§, Dominique Bernard*, Michael D. Cahalan‡¶, K. George Chandy‡, and Evelyne Be´ raud* *Laboratoire d’Immunologie, Faculte´deMe´ decine, 13385 Marseille, France; ‡Department of Physiology and Biophysics, University of California, Irvine, CA 92697; and §Bachem Bioscience, King of Prussia, PA 19406 Communicated by Clay M. Armstrong, University of Pennsylvania School of Medicine, Philadelphia, PA, September 18, 2001 (received for review June 6, 2001) Adoptive transfer experimental autoimmune encephalomyelitis Kv1.3 is found in the hematopoietic lineage, including T and B (AT-EAE), a disease resembling multiple sclerosis, is induced in rats lymphocytes, platelets and megakaryocytes, and microglia, by myelin basic protein (MBP)-activated CD4؉ T lymphocytes. By whereas the distribution of IKCa1 is broader, including a variety patch-clamp analysis, encephalitogenic rat T cells stimulated re- of peripheral tissues and cell types (9, 12, 13). Kv1.3 is the peatedly in vitro expressed a unique channel phenotype (‘‘chron- primary regulator of Ca2ϩ signaling in human quiescent T cells. ically activated’’) with large numbers of Kv1.3 voltage-gated chan- Selective blockade of Kv1.3, but not IKCa1, suppresses mitogen- ؉ ؉ nels (Ϸ1500 per cell) and small numbers of IKCa1 Ca2 -activated K stimulated cytokine production and proliferation of these cells in channels (Ϸ50–120 per cell). In contrast, resting T cells displayed vitro (14–17) and the delayed type hypersensitivity response in 0–10 Kv1.3 and 10–20 IKCa1 channels per cell (‘‘quiescent’’ phe- vivo (6, 18, 19). Transcriptional up-regulation during mitogenesis notype), whereas T cells stimulated once or twice expressed Ϸ200 results in IKCa1 becoming the main regulator of Ca2ϩ signaling Kv1.3 and Ϸ350 IKCa1 channels per cell (‘‘acutely activated’’ phe- in human activated lymphocytes, and selective IKCa1 blockade notype). Consistent with their channel phenotype, [3H]thymidine suppresses cytokine production and mitogenesis of preactivated incorporation by MBP-stimulated chronically activated T cells was cells (17, 20, 21). Because of their important role in T cell suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and activation (9), adhesion, and migration (22), and their function- by an analog (ShK-Dap22) engineered to be highly specific for ally restricted distribution, both channels are widely regarded as Kv1.3, but not by a selective IKCa1 blocker (TRAM-34). The com- novel therapeutic targets. bination of ShK-Dap22 and TRAM-34 enhanced the suppression of Kaliotoxin (KTX), a peptide from scorpion venom that blocks MBP-stimulated T cell proliferation. Based on these in vitro results, -؉ the lymphocyte Kv1.3 and the neuronal Kv1.1 channels, ame we assessed the efficacy of K channel blockers in AT-EAE. Specific liorates the symptoms of adoptive EAE in rats (6). Its thera- and simultaneous blockade of the T cell channels by ShK or by a peutic effect may be due to immunosuppression via Kv1.3 22 combination of ShK-Dap plus TRAM-34 prevented lethal AT-EAE. blockade, or to enhanced nerve conduction via blockade of 22 Blockade of Kv1.3 alone with ShK-Dap , but not of IKCa1 with Kv1.1, or to a combination of both. In the present study, we TRAM-34, was also effective. When administered after the onset of tested whether selective in vivo blockade of the T cell Kϩ 22 symptoms, ShK or the combination of ShK-Dap plus TRAM-34 channels could prevent and treat adoptive EAE in Lewis rats. greatly ameliorated the clinical course of both moderate and ShK, a structurally defined 35-aa polypeptide from the sea severe AT-EAE. We conclude that selective targeting of Kv1.3, anemone Stichodactyla helianthus, was used to inhibit both Kv1.3 alone or with IKCa1, may provide an effective new mode of ϭ ϭ (Kd 11 pM) and IKCa1 (Kd 20 nM). However, this therapy for multiple sclerosis. ϭ ϭ polypeptide also blocks Kv1.1 (Kd 16 pM), Kv1.4 (Kd 314 ϭ pM), and Kv1.6 (Kd 160 pM). Based on sequence differences ultiple sclerosis (MS), a chronic inflammatory autoim- in these channels, we previously engineered ShK-Dap22 to be a ϭ Mmune disease of the central nervous system that commonly highly potent (Kd 20–50 pM) and specific (16, 17, 23) inhibitor affects young adults, is characterized by demyelination and of Kv1.3. We used ShK-Dap22 to selectively target Kv1.3 and the ϭ axonal damage resulting in disabling neurologic deficits (1). clotrimazole analog TRAM-34 (Kd 20 nM) as a selective Experimental autoimmune encephalomyelitis (EAE) mimics blocker of IKCa1 (21). By whole-cell patch-clamp, we demon- many of the clinical and pathological features of MS and is widely strate that rat T cell lines or clones that have been repeatedly used as a model for the human disease (2–4). The central role stimulated with MBP or other antigens exhibit a unique Kϩ of T lymphocytes in pathogenesis is demonstrated by the induc- channel phenotype different from that of normal quiescent or tion of adoptive transfer (AT)-EAE in naive animals after ϩ acutely activated rat T cells. Guided by these patch clamp results adoptive transfer of myelin basic protein (MBP)-activated CD4 and by data from in vitro proliferation assays, we have success- T cells (2, 3, 5–7). Strategies designed to selectively suppress the fully prevented lethal AT-EAE and significantly ameliorated the function of autoreactive T cells represent a potential therapeutic disease when starting treatment after onset of symptoms. approach for MS. Optimal T cell activation requires several hours of calcium ϩ entry from the external milieu through Ca2 release-activated Abbreviations: MBP, myelin basic protein; EAE, experimental autoimmune encephalomy- ϩ ϩ 2ϩ ϩ Ca2 (crac) channels (8–10). Ca2 influx depolarizes the mem- elitis; MS, multiple sclerosis; KCa,Ca -activated K channel; Kv, voltage-gated potassium channel; [3H]TdR, tritiated thymidine; Mt, Mycobacterium tuberculosis; PPD, purified brane and dissipates the electrochemical gradient required for protein derivative of tuberculin; AT, adoptive transfer; KTX, kaliotoxin; MgTX, margatoxin; 2ϩ ϩ further Ca entry. Two K channels, Kv1.3 and IKCa1, provide ChTX, charybdotoxin; TRAM-34, [1-(2-chlorophenyl)diphenylmethyl]-1H-pyrazole. 2ϩ the counterbalancing cation efflux necessary for sustained Ca †C.B. and H.W. contributed equally to this work. influx (10). Kv1.3, a voltage-gated channel, opens in response to ¶To whom reprint requests should be addressed. E-mail: [email protected]. membrane depolarization and maintains the resting membrane The publication costs of this article were defrayed in part by page charge payment. This potential, whereas IKCa1 opens in response to an increase in article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. ϩ cytosolic Ca2 and hyperpolarizes the membrane potential (11). §1734 solely to indicate this fact. 13942–13947 ͉ PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.241497298 Downloaded by guest on September 28, 2021 Materials and Methods continuously present during both phases. PAS T cells were Animals. Female inbred Lewis rats 8–12 weeks of age and guinea stimulated with MBP for 48 h in the presence or absence of 22 pigs were purchased from Harlan Breeding Laboratories (Gan- channel blockers (100 nM ShK, 100 nM ShK-Dap , 500 nM 22 ϩ nat, France). TRAM-34, or 100 nM ShK-Dap 500 nM TRAM-34) or PBS. Cells were washed, viability was assessed by trypan blue dye ϫ 6 Reagents. ShK, ShK-Dap22, margatoxin (MgTX), KTX, and exclusion, and 4–5.5 10 viable cells in 1 ml RPMI medium charybdotoxin (ChTX) were from Bachem (King of Prussia, PA) 1640 were transferred i.p. into four treatment groups of synge- ϩ or from Alomone Laboratories (Jerusalem, Israel). Synthesis of neic rats and into a saline (PBS 2% Lewis rat serum) control group. Soon after, the four groups received i.p. injections of the TRAM-34 ([1-(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) ϩ was previously described (21). Tetraethylammonium, clotrim- same K channel blocker(s) used during the sensitization acti- ␮ 22 ␮ ͞ azole, PHA, and Con A were purchased from Sigma, purified vation step: 16 g ShK or ShK-Dap in 0.5 ml saline (80 g kg), or 34 ␮g TRAM-34 (170 ␮g͞kg) in 0.1 ml of a 50:50 mixture of protein derivative of tuberculin (PPD) from Statens Serum 22 Institut (Copenhagen), complete Freund’s adjuvant and Myco- PBS:DMSO or alcohol or a combination of ShK-Dap and bacterium tuberculosis H37 Ra (Mt) from Difco, and anti-CD4 TRAM-34. The protocol was continued with three more injec- Ab from Serotec. MBP was extracted from frozen guinea pig tions at 4-hr intervals on day 0, three injections on day 1, and two central nervous system (24), purified by C18 reverse-phase injections on days 2–5. Control animals received saline injections HPLC (Millipore͞Waters system), and purity was assessed by with the same protocol. Rats were weighed and observed daily. electrospray mass spectrometry. For the treatment trials, blockers were administered exclu- sively in vivo and after the onset of disease. After the sensitiza- ϫ 6 Cells and Cell Lines. PAS T cells, MHC class II-restricted CD4ϩ T tion step (cells in vitro activated with MBP for 48 h), 3–5 10 cells, are encephalitogenic in vivo and produce limited demyeli- viable PAS T were injected i.p. into syngeneic rats. Because encephalitogenic potency varied from batch to batch, we ad- nation (25, 26). PAS and other MBP-specific T cell lines and justed the number of cells transferred in each experiment to clones were previously established from MBP-primed rat lymph induce disease of differing severity.

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