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~~;~; ~ELSEVIER Immunopharmacology 48 (2000) 51-63 ;.~

www.elsevier.com/locate/immpharm "'

K+ channel-blocking alkoxypsoralens inhibit the immune +'j~

response of encephalitogenic T line cells and lymphocytes from t;c

Lewis rats challenged für experimental auto immune

encephalomyelitis

VIf Strauss a, Kirsten Wissel a, Stefan Jung b, Heike Wulff c, Wolfram Hänsel c,Jie Zhu d, Arndt Rolfs a, Eilhard Mix a, "

a Department of Neurology, University of Rostock, P.O. Box 100888, Gehlsheimer Str. 20, D-18147 Rostock, Gennanyb Department of Neurology, Julius-Maximilians-Universität, Josef-Schneider-Str. 11, D-97080 Wurzburg, Gennany

C Pharmaceutical Institute, Christian-Albrechts-Universität, Gutenbergstr. 76178, D-24118 Kiel, Gennanyd Division of Neurology, Karolinska Institute, Huddinge Unu'ersity Hospital, S-14186 Huddinge, Sweden

Received 18 August 1999; received in revised form 6 January 2000; accepted 6 January 2000

Abstract

Alkoxypsoralens, known as DNA photomodifying agents, have been shown to block voltage-dependent K+ channels(Kv) as weIl as to alleviate functional deficits in certain multiple sclerosis (MS) patients in a manner similar to4-aminopyridine. Since Kv channel blockers are known to inhibit T cell-mediated immune responses both in vitra and in'vivo, we investigated the effects of three alkoxypsoralens, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and5,8-diethoxypsoralen (H37), on the following parameters: (1) whole-cell K+ currents of encephalitogenic, myelin basicprotein-specific memory T cellline cells (MBP-TCLC) derived from Lewis rats as measured by patch-clamp technique, (2)proliferation of MBP-TCLC and lymph node cells (LNC) from Lewis rats challenged für experimental autoimmuneencephalomyelitis (EAE) by immunisation with spinal cord homogenate as measured by 3H-thymidine incorporation, (3)interferon-')' (IFN-')') secretion of MBP-TCLC as measured by EUSA, and (4) IFN-')' gene expression of LNC as measuredby quantitative reverse transcription polymerase chain reaction (RT -PCR) with EUSA-detection. The examined alkoxypso-ralens exhibited suppressive effects on the measured parameters with the same sequence of efficacy: H37 > 5-MOP > 8-MOP.We, therefore, conclude that Kv channel-blocking alkoxypsoralens interfere with voltage-controlled signal transduction inlymphocytes and might thereby suppress immune responses in autoimmune diseases of the central nervous system and most

Abbreviations: APC, antigen presenting cells; CoDA, concanavalin A; DMSO, dimethylsulfoxide; EAE, experimental autoimmuneencephalomyelitis; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; GP-MBP, guinea-pig myelin basic protein;GP-SCM, guinea-pig spinal cord myelin; 1FN--y, interferon--y; Kv, voltage-dependent K+ channel; LNC, lymph node cells; MBP-TCLC,myelin basic protein-specific T cell1ine cel1s; MOP, methoxypsoralen; MS, multiple sclerosis; PCR, polymerase chain reaction; PUVA,psoralen plus ultraviolet A radiation; RT-PCR, reverse transcription polymerase chain reaction: TUNEL, terminal transferase ddUTP nickend labeling. Corresponding author. Tel.: +49-381-4949598; fax: +49-381-4949598.

E-mail address: eilhard.mix@med.uni-rostock.de (E. Mix).

0162-3109/00/$ - see front matter ~ 2000 Elsevier Science B.V. All rights reserved.PU: SOI62-3109(00)00177-6 ..

52 U. Srrauss er al. / Immunophannacology 48 (2000) 51-63 "c~

~.~

likely also in other autoimmune disorders. Thus, älkoxypsoralens, especially the non-phototoxic substance H37, are newcandidates für further studies on K+ channel blocking immunosuppressive drugs. The agents may exert a dual beneficialeffect on demyelinating diseases like MS, because they could attenuate the inflammatory process and improve axonalconductivity. @2000 Elsevier Science B.V. All rights reserved.

Keywords: AlkoxypsoraJens; K+ channels; Lymphocyte proliferation; Interferon--y; Experimental autoimmune encephalomyelitis

1. Introduction soralen (5-MOP) and 8-methoxypsoralen (8-MOP)are planar bifunctional photoactivatable agents, which

The crucial role of voltage-gated K + channels are of wide clinical use in psoralen plus ultraviolet A

in the regulation of physiological functions in exci- radiation (PUV A) therapy of psoriasis, vitiligo, my-table as weil as non-excitable cells is weil known cosis fungoides and for cutaneous T celllymphoma.(Rille, 1992; Chandy and Gutman, 1995). Non-exci- The antiproliferative therapeutic effect as weil as thetable cells such as T lymphocytes are important for photomutagenic and photocarcinogenic side-effectsthe mediation of a number of autoimmune diseases, of psoralens are due to a combination of photodam-including multiple sclerosis (MS), which can be age to DNA (Averbeck, 1989), proteins and mem- f:!studied in an animal model, i.e. experimental brane lipids (Beijersbergen van Henegouwen et al., Iiautoimmune encephalomyelitis (EAE) (Martin and 1989; Zarebska, 1994). Recently, the K+ channel ~McFarland, 1997). The opening of voltage-depen- blocking activity of alkoxypsoralens has been sepa- : ~dent K+ channels (Kv), especially the Shaker-related rated from their inherent phototoxicity by synthesis-K+ channel Kv1.3, represents an early event in the of new psoralen derivatives like 5,8-diethoxypsora- ~~

""

activation of T lymphocytes in response to mitogenic len (H37) (Wulff et al., 1998b). This enabled us to ~~~

(DeCoursey et al., 1984; Cahalan and Chandy, 1997) investigate the immunomodulatory and K+ channel rz~

and antigenic (Strauss et al., 1998) stimulation. 4- blocking properties of a non-phototoxic psoralen ["aminopyridine, tetraethylammonium chloride, qui- (H37). In contrast to 5-MOP (Zajde1a and Bisagni, fc:,

nine, and verapami1 block Kv1.3 and in parallel 1981), the 10ng-term therapeutic use of H37 shou1d r

inhibit T-1ymphocyte activation (Chandy et al., 1984; not be limited by any phototoxic or photomutage~c ! ~'-Cahalan et al., 1985). Michne et al. (1995) reported side-effects.on the synthesis and bio10gical characterisation of The present study aims to exp10re the ability of :,:4-(alkylamino)-l,4-dihydroquinolines as new potent differently substituted alkoxypsoralens (5-MOP, 8-Kv1.3 blockers on the basis of inhibition of charyb- MOP and H37) to inhibit K+ currents in encephali-dotoxin binding and inhibition of whole-cell Kv1.3 togenic T 1ymphocytes and to study in parallel theirtype currents in human Jurkat T 1ymphocytes in vitro effects on the functional activity of T 1ymphocytes inwith the aim of further development of anti-in- EAE of Lewis rats. The T cell activity is measured inflammatory drugs. Koo et al. (1997) showed that EAE lymph node cells (LNC) in two in vitro sys-margatoxin, a selective peptidic inhibitor of Kv1.3, tems, which reflect their ability to induce inflamma-suppresses immune responses in vivo. Thus, Kv1.3 tory demyelination in vivo: (1) proliferation in re-may be considered as a novel pharmacological target sponse to antigenic stimulation by a myelin prepara-for immunosuppressive therapy. tion and to mitogenic stimulation by concanavalin A

In a study on homomeric Kv channels expressed (ConA) as a measure for polyclonal expansion ofin mammalian cells, certain alkoxypsoralens were myelin reactive and bystander cells, and (2) inter-found effectively to block a number of Shaker-re- feron-'"'{ (IFN-'"'{) gene expression as a parameter oflated K+ channels, including Kv1.3 (Wulff et al., pathogenically important proinflammatory cytokines1998a). Therefore, alkoxypsoralens could have im- in EAE (Renno et al., 1994; Issazadeh et al., 1995;munosuppressive effects on autoreactive T lympho- McCombe et al., 1998) and MS (Olsson et al., 1990;cytes, which are independent from their well-known Woodroofe and Cuzner, 1993; Link, 1998). In orderphototoxicity. Alkoxypsoralens like 5-methoxyp- to compare the immunomodulatory effects of the

U. Strauss et al./ lmmunophannacology 48 (2000) 51-63 53 ',c"r-:~-,

alkoxypsoralens with their K + channel blocking ac- - ing bad been freshly restimulated tor 3 darg with itivity, T celllines derived from EAE Lewis rats with GP-MBP and APC and separated from APC by !

defmed encephalitogenicity have been used tor Ficoll gradients. They were cultured in 96-well mi- [whole-cell patch-clamp recordings of K+ currents crotiter plates in RPMI 1640 medium supplemented t',-

(Strauss et al., 1998). with 10% fetal calf serum (FCS), glutamax II, 1 % ~-sodium pyruvate, 100 U Iml penicillin, 100 ~g/ml t

streptomycin, 10 - 5 M mercaptoethanol and 10

2. Material and methods ~g/ml indomethacin (all reagents from Gibco) at37°C and 6% CO2 in a humidified atmosphere. Ten

2.1. Alkoxypsoralens percent of interleukin-2 containing supematant fromConA-activated syngeneic spleen cells was added to

5-MOP and 8-MOP were purchased from Sigma the medium as essential growth factor.(St. Lewis, USA). H37 was prepared as described byWulff et al. (1998b) (Fig. 1). Briefly, demethylationof 8-MOP with magnesium iodide yielded xantho- 2.3. Patch-clamp measurements on MBP-TCLC

toxol, which after oxidation with chromium trioxide,gave the corresponding quinone. Reduction of the Patch-clamp experiments were carried out onquinone with zinc dust in diluted HCl provided the MBP- TCLC at room temperature (20°C-24°C). Patchhydroquinone 5,8-dihydroxypsoralen, which was re- pipettes were prepared from thick-walled glass capil-acted with diethyl sulfate in the presence of potas- laries (Corning #1010; Wor1d Precision Instruments,sium carbonate to give H37. Stock solutions of Sarasota, USA), which were pulled in two stages onpsoralens were prepared at 2 X 10-2 M in dimethyl- a PUL-100 puller (World Precision Instruments) andsulfoxide (DMSO; Sigma). Dilutions were made in fIre polished. The pipettes bad a resistance in the0.5% DMSO in RPMI 1640 (Gibco BRL, Life Tech- range of 1-3 Mo' with the solutions described be-nologies, Paisley, UK). low. For the experiments carried out in this study,

the whole-cell configuration of the patch-clamp tech-2.2. Cultures of encephalitogenic T line cells [myelin nique (Hamill et al., 1981) was used. Access resis-basic protein-specific T cellline cells (MBP-TCLC)] tance was checked regularly during the course of the.. experiments to ensure stable registration conditions. 1 ; c

Encephalitogenic T cell lines have been estab-. Membrane currents were evoked with depolarisinglished from Lewis rats immunised with guinea-pig pulses to + 30 mV with a duration of 400 ms from amyelinbasicprotein (GP-MBP) in completeFreund's holding potential of -80 mV, which were appliedadjuvant as described previously (Jung et al., 1992). every 50 s. All recordings were made with an Ax-Briefly, popliteal lymph node-derived CD4 + MBP- opatch 200A amplifier (Axon Instruments, FosterTCLC were propagated by repeated in vitro restimu- City, USA). The data were filtered at 10 kHz andlation with GP-MBP and irradiated syngeneic thYT sampled at 1 kHz. The amplitudes of the whole-cellmocytes as antigen presenting cells (APC) every 7-8 currents were measured in most cases when thedarg. The MBP- TCLC used tor patch-clamp record- current reached a maximum before inactivation starts

and, therefore, are expressed as peak current ampli-tudes. The currents measured in MBP- TCLC showed

o"'~ O...~Hs a rundown, i.e. their amplitude decreased and N-typef""T::::::",~ i""".)"~ <W ~ ~ inactivation accelerated with time after establishing-O""'lAo~ \0.J~,,~ 0 ~ I the whole-cell configuration. Only cells with a slow

0, 0 0 0 0 0 0 linear rundown were used tor analysis and the exper-

CH] 'C2Hs iment was finished when the current was reduced by8-MOP 5-MOP 1137 more than 25% of its initial value. The acceleration

Fig. 1. Structures of the alkoxypsoralens 5-MOP, 8-MOP, and of the inactivation was generally less affected by theH37. rundown. The linear rundown with time was cor-

54 U. Strauss et al./Tmmunophannacology 48 (2000) 51-63 r~- ..'y

f';rected tor during data analysis. 1t should be men- crude myelin preparation was dialysed against water f'tioned that it was necessary to wait up to 15 min tor 24-48 h and lyophilised. Resulting GP-SCM "

after establishing the whole-cell configuration to get were added to LNC suspensions to obtain a fmalstable currents. The pipette solution contained (in concentration of 0.2 mg/mI.mM): KF 140, CaClz 1, MgClz 2, KCI 3, EGTA 11, For mitogenic stimulation, ConA (Sigma) wasHEPES 10, pH 7.2. The composition of the solution added at a final concentration of 4 IJ.g/ml.was modified according to DeCoursey et al. (1984)

Iand Oleson et al. (1993) in order to establish optimal 2.5. Proliferation assaysrecording conditions. The bath solution contained (in '~mM): NaCI 150, KCI 4.5, CaClz 2.5, MgClz 1, LNC of EAE rats were cultivated tor 72 h in the l'-:HEPES 10, pH 7.4, 290 mOsm. This bath solution presence or absence of the indicated stimulants (GP- -

was continuously perfused into the experimental SCM or ConA) and psoralens. Two independentchamber by use of a syringe pump under non-pulsa- series of experiments were performed with 5-MOP , 1tile and low (45 ml/h) constant flow conditions. All and 8-MOP (seven animals each) and H37 (three ' ~

~;~i test substances were added into the perfusion line by animals), respectively. Ten hours before termination ::~

a second syringe pump working at 1:100 of the of the culture, cells were pulsed with 3H-methyl- ::~pumping rate of the fIrst pump. A complete ex- thymidine (10 IJ.I, 100 IJ.Ci/ml; Amersham, Little r,:change of the bath solution in the experimental Chalfont, UK). Cells were then harvested onto glasschamber with this syringe pump-system took 25 s. fibre fllter strips (Skatron, Lierbyen, Norway) with a

multi-channel semiautomated harvesting device2.4. Preparation and cultivation oi EAE LNC (Titertek, Skatron) followed by repeated washings in

distilled water. The radioactivity incorporated intoLewis rats were immunised by injection into each cellular DNA was counted on a beta scintillation

bind footpad of 0.1 ml inoculum containing 1 mg counter. The mean counts per minute (cpm) wereMycobacterium tuberculosis (ST 37 RA, Difco, De- calculated from triplicate weils.troit, USA), 0.05 ml saline, 0.05 ml Freund's incom- Activation of the MBP-TCLC was measured inpIere adjuvant (Difco) and 25 mg homogenised flat-bottomed 96-well microtiter plates. 1.75 X 104guinea-pig spinal cord. Rats were killed at days responder T cells and 1.25 X 105 irradiated syn-7 - 21 after immunisation. Suspensions of popliteal geneic spleen cells were seeded in 100 IJ.I of restimu-

and inguinal LNC obtained by crushing through lation medium per weil. GP-MBP was used at asterile wire meshes were prepared in sterile phos- concentration of 20 IJ.g/ml and ConA at a concen-phate buffered saline, pH 7.4 (Gibco). Cells were tration of 2 IJ.g/ml. 10 IJ.I of RPMI/DMSO orwashed twice and resuspended in RPMI with gluta- indicated concentrations of 8-MOP, 5-MOP, or H37max ll, 50 IU /ml penicillin, 60 IJ.g/ml strepto- in RPMI/DMSO Were added in a volume of 10 IJ.I.mycin and 5% FCS (all reagents from Gibco). Tripli- 3H-methyl-thymidine incorporation was measured tcate aliquots (200 IJ.I) of LNC suspensions were during the last 16 h of the 72-h culture as described ~

added to flat-bottomed 96-well microtiter plates above. l(Nunc, Copenhagen, Denmark) at a cell density of ~c;;2 X 106 cells/ml medium. 2.6. Generation oi culture supematants from MBp. f

For antigenic stimulation, guinea-pig spinal cord TCLC and determination oi IFN-y by enzyme.;linked f,

myelin (GP-SCM) was used. The GP-SCM was pre- immunosorbent assay (ELISA) :

pared as described elsewhere (Norton and Poduslo,1973). Briefly, guinea-pig spinal cords were crushed 6.6 X 10sMBP-TCLC and 5.0 X 106 irradiatedto small pieces in liquid nitrogen and homogenised syngeneic spleen cells per 2 ml of restimulationin 0.3 M sucrose. The homogenates were overlaid medium were seeded in 24-well Nunclon dishesonto 0.85 M sucrose and centrifuged tor 1 h at 4°C. (Nunc, Roskilde, Denmark). A volume of 200 IJ.IThe interfaces were resuspended in water dilution RPMI/DMSO, or 8-MOP, 5-MOP, or H37 (each in1:10 and centrifuged twice at 4°C tor 20 min. The 200 IJ.I RPMI/DMSO) were added per weIl and

U. Strauss et aLl Immunopharmacology48 (2000) 51-63 55

cells were activated by GP-MBP (20 ,...g/ml). Cul- the six-base pair deletion, either standard or wild-typetuTe supernatants were collected 72 h later and frozen - amplicons. A digoxigenin-labelled detection probe,

in two aliquots at - 80°C. Concentrations of IFN-'Y which was complementary to sequences common to

in the supernatants were determined by the rat IFN-'Y the standard and wild-type amplicons, was used to fDuo Set from Genzyme (Rüsselsheim, Germany) in detect the immobilised capture probe/amplicon hy- r

triplicate analysis as recommended by the manufac- brids. The bound digoxigenin was quantified with an ~-"

tuTeT. an~-digoxigenin alkaline phosphatase. conjugate ,t,,;(SIgma) and 100 ,...1 substrate, para-mtrophenyl- l

2.7. Quantitative reverse transcription polymerase phosphate (10 mg in 11 ml). Optical density was fchain reaction (RT -PCR) tor measurement of IFN-'}' measured at 405 nm in a 96-channel EL1SA readergene expression of EAE LNC after 60 min incubation at 37°C. From a calibration

curve derived from different mixtures of knownLNC of EAE rats were cultivated as tor the numbers of standard and wild-type amplicons, the

proliferation assay. After 72 h of incubation, total number of wild-type copies present in the originalRNA was isolated by acid phenol/chloroform ex- cell cultures were estimated exact1y.traction (Chomczynski and Sacchi, 1987) (RNAzolB; AGS, Heidelberg, Germany) and coprepared with 2.8. Statistical analysisa mutagenised internal RNA standard. A six-base . , . .pair deleted IFN-'Y cRNA served as an internal stan- Two-tailed Student ~ t-test tor .pmed.obser:ationsdard fragment. 1t was constructed according to Bauer was used to analyse ~erences m proliferation andet al. (1996) in order to get a lang internal standard IFN-'Y gene expressIon between untreated andfragment, differing from the IFN-')' wild-type mRNA alkoxyps~rale~-treated cells as weIl as between cellsonly in a few base pairs. This mutagenising of the ~ea~d Wlth different alkoxypsoralens. The level ofwild type is necessary to differentiate it from the sIgnificance was set to p < 0.05.

standard by liquid phase hybridisation. Up to 1 ,...gisolated total RNA was reversely transcribed with 1 3. Results,...g random hexamers and 100 U superscript (Gibco).Polymerase chain reaction (PCR) of 50 ng wild-type 3.1. Alkoxypsoralens inhibit whole-cell K + currentscDNA, together with a given amount of standard of MBP-TCLC f

cRNA was amplified in a 25-,...1 PCR assay [30cycles, 5 min 94°C (1 min 94°C, 1 min 48°C-72°C, . To investigate the effect of alkoxypsoralens on

1 min 72°C; X 30), 5 min 72°C, 1.0 U Taq poly- whole-cell K+ currents of MBP-TCLC, the sub-merase, 4 pmol of the two primers (5'-GGAG- stances 8-MOP, 5-MOP and H37 were added to theGAACTGGCAAAAGGAC-3' and 5'-CCAGAAT- bath solution of the patch-clamp device after stahleCAGCACCGACTCC-3'), 10 mM Tris HCl, 50 mM membrane seals bad been achieved and control cur-KCl, 2.5 mM MgClz, 100 ,...M dNTP] in a thermo- rents were recorded. For this purpose, the membranecycler PE 9600 (Perkin-Elmer, Foster City, USA). potential was clamped every 50 s from a holdingThe design of the left primer as a semijunction potential of - 80 mV up to + 30 mV tor 400 ms inprimer served to avoid DNA coamplification (Moore order to induce a transient current. Fig. 2 showset al., 1990; Rolfs et al., 1992). Aliquots of the PCR representative examples of the control currents andproducts were quantified in a liquid phase hybridisa- the effects of the tested alkoxypsoralens includingtion/EL1SA system as previously described (Rolfs washout effects. All tested alkoxypsoralens inhibitedet al., 1992). Briefly, specific biotinylated capture the peak current and increased the rate of currentprobes tor standard or wild-type amplicons were decline during the pulse. In both respects, theybound in different wells of a 96-well streptavidin- showed different dose-dependent potency. H37coated microtiter plate (Microcoat, Penzberg, Ger- tumed out to be the most potent blocker of K+many). In the following hybridisation step, the cap- whole-cell peak currents with an ECso of 6.7:!:: 0.1tuTe probes recognise and bind specifically, based on ,...M tor peak current inhibition, followed by 5-MOP

,

,...r

rr,f"

i:~

;k~- ~

56 U. Strauss et al./ Jmmunophannacology 48 (2000) 51-63 tff",

H37f-600 control

2xl0-6 Mwashout

-. 5xl0-6 M<I:: 400 7.5xl0-6 M0.. 10-5 M

Q 1.5xl0-5 M~ 200 2xl0-5 M

0

0 500t (ms) -=

1;'1 control 5-MOP -. .'

800 5xlO-6 Mwashout10-5M

--- 600 2xlO-5 M !:::~ "-xlO-5 M C

0... 400 5xl0-5 M-.;;;;; 7.5xlO-5 M~ 200

0 f

,-200 ~'

200 300 0t (ms)

600 8-MOP ~control and washout7.5xl0-5 M

-. 400 10-4 M<I:: 1.5xl0-4 M.E:; 2xl0-4 M~ 200 3xl0-4 M

0

100 200 300 500t (ms)

Fig. 2. Inhibitory effect of the alkoxypsoralens H37, 5-MOP, and 8-MOP on whole-cell K+ currents of MBP-TCLC as measured by thepatch-clamp technique. The traces are recorded after reaching a steady state at each concentration. Only pipette capacitance wascompensated. .

Ii

t;

,,U. Strauss et al./lmmunopharmacology 48 (2000) 51-63 57 r

..~

F-'(ECso = 28.1 :I: 1.0 IJ.M) and 8-MOP (ECso = 212.6 mum inhibitory concentrations were as follows: H37:, r7

:I: 10.5 IJ.M) (Fig. 3A). Time constants of N-type -ECso = 5.3:1: 0.3 IJ.M, 5-MOP: 19.5:1: 1.6 IJ.M, 8- '[(fast) inactivation were achieved by a single expo- MOP: 242:1: 24 IJ.M (Fig. 3B). The blocking effects ;-

nential fit of the decaying phase of the current and were fully developed after 90 s and almost com- [. ,divided by the rundown corrected control. The result- pletely reversible within 150 s after the onset of r

ing ratlos represent a measure of the effect of the washing-out. Because of the high lipophilicity of the ldifferent alkoxypsoralens on inactivation. Half maxi- test substances, DMSO was used as a cosolvent. ,j~

Addition of DMSO alone up to a concentration of ' ,"

2%, which is the maximum concentration applied ",.

together with the drugs, did not exert significant :~

A 100 [;;] H37 effects wh~n evaluated statistically. Ho~ever, sev- :~

. S-MOP eral recordings showed up to 10% reductlon of peak ?]~80 T 8-MOP currents and acceleration of inactivation time con- :,~

'Ö' stants compared to controls (data not shown). This is r'~ 60 most likely the reason why, in same cases, the ,.

§ highest concentration of the tested substances that:-2 40 had still no effect was not exactly determinable.~ Therefore, the fitting curves seen in Fig. 3A and B.1 do not asymptote exactly to the values zero and one,

20 .I .

respective y. .

0 3.2. Alkoxypsoralens inhibit proliferation 01 EAE10 10.' 10-4 LNC

concentration (M)B ~ H37 In order to investigate the effect of alkoxypso-

1.0 . S-MOP ralens on lymphocyte proliferation, LNC of rats im-T 8.MOP munized with GP-SCM were exposed to different

.g fJ 0.8 concentrations of the three tested psoralen deriva-

.~ j tives 8-MOP, 5-MOP and H37. The concentration~ ~ 0.6 showing the highest discrimination of the antiprolif - ,

~ u 0.4 erative effect between the three substances was 2 X_,.,§ j 10-s M. This concentration was applied in sevene 0.2 experiments tor testing of 5-MOP and 8-MOP and in

three experiments tor testing of H37. At this concen- l.0.0 tration, the drugs did not exert cytotoxic effects on

I10-6 10-'. 10-4 LNC, neither in the trypan bIlle exclusion test torconcentration (M) detection of necrosis, nOT in the terminal transferase ',.,

Fig. 3. Dose-response relationships tor suppression of K+ current ddUTP nick end labeling (TUNEL) assay tor detec-on MBP-TCLC by alkoxypsoralens (~ H37, 0 5-MOP, "7 tion of apoptosis (unpublished data).8-MOP). Each point represents the mean value (.+.SEM) of 3 to As shown in Fig. 4, H37 and 5-MOP exhibited10 experiments. Data in A and B were fi~ed visually. (A) Effect higher suppressive effects than 8-MOP. The extenton peak currents. The best fit tor the different alkoxypsoralens .. . . . . '. .wasreachedbyuseofHill'sequationY=Ymaxxxb/(xb+EC~o) o~ inhib.ltlOn was similar for.LNC ~lthOUt ~ Vl~O r~

resulting in the following values of ECso: 6.7.+.0.1 IJ.M tor H37, stImulation and tor LNC Wlth antlgen-specific m28.1'+'1.0 IJ.M tor 5-MOP, and 212.6.+.10.5 IJ.M tor 8-MOP. (B) vitra stimulation by GP-SCM ranging between 60%Effect on inactivation time constants quantified as described in and 70% tor H37 and 5-MOP, and 30% and 40% torSection 3. The best fit tor the di!ferent alkoxypsoralens was 8-MOP, respectively. ConA-stimulated cells werereached by use ofBoltzmann's equation Y = Ymax x{1 +exp[(x - .. . .1Cso)/dxD resulting in the following value of ECso: 5.3.+.0.3 inhiblted to a lower extent than GP-SCM-stImulatedIJ.M tor H37, 19.5.+.1.6 IJ.M tor 5-MOP, and 242.+.24 IJ.M far cells by an three drugs. However, H37 also exhibited8-MOP. the highest inhibitory potency (54%) in these cells,

~

" ;c

,,~

l:i,,

;F~- ~

58 U. Strauss et al./ Immunophannacology 48 (2000) 51-63 -.

A B 350000 ORPMilDMSO20000 e

---5 300000 '.MOP

"8-OIOPQ,~ 15000 2500004) 8RPMi/DMSO c;

~ 200000 CHI'_) ;c:'-- . 8M3' cQ,:= . ':~ 10000 . 150000.- . .

~

.~ 100000

..s 5000

:.:: 50000'"

0 0

no antigen GP-SCM ConA

Fig. 4. Inhibitory effect of aIkoxypsoralens at the concentration of 2 x 10-5 M on proliferation of LNC from guinea-pig spinal

cord-immunised Lewis rats. (A) Incubation without stimulation (no antigen) and with antigenic stimulation (GP-SCM). (B) Incubation with

mitogenic stimulation (ConA). 5-MOP and 8-MOP were tested in seven different experiments; H37 was tested in aseparate genes of threeexperiments. Therefore, the H37 data refer to aseparate RPMI/DMSO control. Columns represent mean values (:t: SEM). P-values refer tocomparisons between RPMI/DMSO control cells and drug-treated cells: . p < 0.05, . . p < 0.02, . . . p < 0.01.

whereas 8-MOP bad the lowest inhibitory effect 0.5%, which was also used for application of the(16%). All drug effects refer to control cultures with drugs. F!ifi:DMSO at thefinal concentrationof 0.5%, which was l~also used for application of the drugs. The drugs did 3.4. Alkoxypsoralens inhibit proliferation oi MBP- :not show a tendency to higher or tower suppression TCLCduring different phases of the disease, i.e. from day 7to day 21 post-immunisation. The proliferation of MBP-TCLC was inhibited by

the tested alkoxypsoralens to a similar extent and3.3. Alkoxypsoralens inhibit IFN-i' gene expression with a similar order of potency as the proliferation ofoi EAE LNC EAE LNC. Fig. 6 shows representative results of one

out of two experiments. In GP-MBP-stimulatedIn parallel to their inhibition oflymphocyte prolif- MBP-TCLC, H37 bad the highest (75%) inhibitory

eration, suppressive effects on gene expression of the effect, whereas both 5-MOP and 8-MOP were in-proinflammatory cytokine IFN--y was detected for hibitory to tower extents (less than 30%). Data inthe psoralens 5-MOP and 8-MOP in EAE LNC. parentheses refer to the figure. In ConA-stimulatedIFN--y mRNA was measured by quantitative RT- MBP-TCLC, which showed tower proliferative activ-PCR. Fig. 5 shows the higher inhibitory effect of ity compared to GP-MBP-stimulated MBP-TCLC,5-MOP compared to 8-MOP in EAE LNC stimulated H37 was also most effective (85% inhibition), whilewith GP-SCM (5-MOP: 66% versus 8-MOP: 37%, 8-MOP showed the lowest inhibitory effect (35%).p < 0.02) or ConA (5-MOP: 49% versus 8-MOP: MBP- TCLC without antigen or mitogen as weil as21 %, p < 0.05). In unstimulated LNC, the inhibitory APC alone in the presence of MBP or ConA did noteffects were not significant. In LNC from naive exhibit relevant proliferation (226 :i: 43, 36:1:: 3, andLewis rats, IFN--y mRNA could not be detected, 179:1:: 84 cpm, respectively).apart from ConA-stimulated LNC. In these cells, theConA-induced IFN--y gene expression (97:1:: 8 copies 3.5. Alkoxypsoralens suppress IFN-i' production oiof IFN--y mRNA per cell) and the inhibitory effects MBP-TCLCof 5-MOP (69%) and 8-MOP (27%) were similar tothose in EAE LNC. All drug effects refer to control In parallel to the proliferation assay, the IFN--ycultures with DMSO at the final concentration of production of MBP- TCLC was measured in cell

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,-,. 160 .'~- ~~ DRPMI/DMSO ;1;:(.) E5-MOP .'l,'"" * T8. m 8-MOP [cr/) 120 * fQ) I.-CI. l

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Fig. 5. Inhibitory effect of a1koxypsoralens at the concentration of 2 x 10-s Mon IFN-'Y gene expression in LNC from guinea-pig spinal ,~cord-immunised Lewis rats with or without mitogenic (CoDA) or antigenic (GP-SCM) stimulation as measured by quantitative RT-PCR.Columns represent mean values (:f:SEM) of 4 (no antigen and GP-SCM). or 7 (CoDA) different experiments. P-values refer to comparisons. .. ... cbetWeen RPMI/DMSO control cells and drug-treated cells: p < 0.05. P < 0.02. p < 0.01. .;

culture supematants. Fig. 7 shows representative re- erative activity (see Fig. 6). In these cells, H37 badsults of ODe out of two experiments. The highest the highest (67%) and 8-MOP the lowest (34%)IFN--y production was found in the GP-MBP-stimu- inhibitory effect. In ConA-stimulated MBP- TCLC,lated MBP- TCLC, which bad also the highest prolif- only 5-MOP exhibited an inhibitory effect (54%).

,.60000 DRPMl/DMSO"8H37E5MOP

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d0

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no antigen GP-MBP ConA I~ig. ~. Suppression of antigen- and miragen-driven MBP- TCLC activation by a1koxypsoralens. Proliferation of the MBP- TCLC activated by IIrradlated APC and indicated antigen (GP-MBP) or miragen (CoDA) was measured in microtiter wells during the last 16 h of a 72-h ~.

incubation period in the absence or presence of indicated a1koxypsoralens (2 X 10-s M). Bars indicate SD.

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~ 300~

! 200zn~ 100-

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Fig. 7. Alkoxypsoralens suppress IFN-'Y production of antigen or mitogen-activated MBP. TCLC. MBP. TCLC were incubated withirradiated APC in the absence or presence of indicated antigen (GP-MBP) or mitogen (CoDA) in part after addition of indicatedalkoxypsoralens (2 X 10-3 M). IFN-'Y concentrations were determined by ELISA in the 72-h culture supernatants. Bars indicate SD.

MBP-TCLC without antigen or mitogen as weil as (Wulff et al., 1998c). Another mechanism für theAPC alone in the presence of MBP or ConA did not increased rate of current decline during the stimu1at-produce relevant amounts of IFN-'Y (0.3, 0.004, and ing pulses cou1d be a rapid open channel block.1.7 ngjml, respective1y). Since we have previously shown that vo1tage-

gated K + currents were highly corre1ated to the

encephalitogenicity of the same T line ceils (Strauss4. Discussion et al., 1998), and since K+ channe1 blockers have ,

~ been reported to exert immunosuppressive effects in

The present study shows that three alkoxypso- various systems including autoimmune diseasesralens, 5-MOP, 8-MOP and H37, inhibit voltage- (Cahalan and Chandy, 1997; Koo et al., 1997), Dur '..:,igated whole-ceil K+ currents in encephalitogenic T finding prompted us to investigate the effect of the t;:l:jline ceils of Lewis rats. Concerning the mechanism three alkoxypsoralens on the proliferative responseof the observed K+ current block, there is evidence of LNC in EAE-challenged Lewis rats für the fol-für a resemblance to a K+ current block described lowing reasons: (1) EAE is a wide1y used animalfür bretylium tosylate and ß-scorpion toxin 2 by model für studying the autoimmune response and itsGaspar et al. (1995): (1) the drug-induced change in therapeutic manipulation in the nervous systeminactivation rate and (2) the correspondence of (Martin and McFarland, 1997). (2) LNC serve as adose-response relationships für inactivation time source of autoreactive and bystander T ceils, whichconstant decay and decrease in peak current. There- reflect the systemic auto immune response of thefore, it is proposed that the action of the drugs takes organism and which are easily available für in vitro ~

place at a regulatory site of K+ channels on the studies of autoimmunity.intraceilular or extraceilular surface. The drugs are Gur results show that the drug with the highestsupposed to stabilise inactivated conformational K + blocking activity (H37) has the strongest in-states of the pore so that opening becomes less likely hibitory effect on antigen-specific and mitogen-in-and the inactivation rate may increase (Hille, 1992). duced LNC proliferation of GP-SCM-immunised ratsThis hypothesis is consistent with fmdings on rat and the drug with the lowest K + current blockingbasophilic leukemia ceils injected with Kv1.3 cRNA activity (8-MOP) hag the lowest antiproliferative ef-

..

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U. Strauss et al./ 1mmunopharmacology 48 (2000) 51-63 61

fect (Fig. 4). This finding leads us to conclude that cally with CoDA (Fig. 5), argues for an action of 'the suppressive effect of the tested alkoxypsoralens - 5-MOP on voltage-controlled signals of T cell acti-

on LNC may be due to their interference with volt- vation.age-controlled signal transduction in lymphocytes. In addition to the testing on EAE LNC, psoralensThe mechanism of suppression of the voltage-con- have been applied to the encephalitogenic TCLC thattrolled signal transduction relies most probablyon were also used for estimation of K+ current blockinginhibition of Ca + influx (Kerschbaum and Cahalan, properties. The suppressive effects on ConA-stimu-

1999) through the hyperpolarised plasma membrane lated proliferation were similar to EAE LNC (orderof the T lymphocytes (Verheugen and Vijverberg, of efficacy: H37 > 5-MOP > 8-MOP), while 5-MOP1995). This interpretation may particularly apply for exhibited a smaller inhibitory effect on GP-MBP-the alkoxypsoralens H37 and 5-MOP, which have stimulated TCLC than on myelin-stimulated EAEhigh K+ current blocking effects. However, interfer- LNC. In antigen-stimulated TCLC, the suppressionence of psoralens with other signal pathways of T of IFN-')' protein secretion was similar to that ofcell activation is not excluded. An antiproliferative IFN-')' gene expression in EAE LNC, i.e. the ordereffect of 8-MOP has been shown in human blood of inhibitory potency was H37 > 5-MOP > 8-MOP.lymphocytes (Cox et al., 1987), hut the mechanism However, using memory TCLC and analysing IFN-')'of action has not been unravelled so far. On the other at the protein level, a suppression by H37 and 8-MOPhand, co-mitogenic effects of psoralen on human was not observed. This might be explained by differ-blood lymphocytes have also been found (Arslan et ent inhibitory influences of H37 and 8-MOP on theal., 1989). various activation pathways in antigenically and mi-

In the presence of ultraviolet radiation, 8-MOP togenically stimulated TCLC, respectively.and 5-MOP, hut not H37, intercalate by covalent Alkoxypsoralens exhibit a relatively low selectiv-binding into double-stranded nucleic acids, espe- ity tor voltage-gated K + channels within the Shaker

cially with high A- T content, as measured by a shift family and the blocking potency depends on theof the thermal transition temperature (Wulff et al., substitution pattern. Compounds hearing alkoxy1998b). Since H37 is lacking this photobinding prop- groups in the 5-position and both in 5- and 8-posi-erty, it may be the drug of the highest practical tions are much more potent than compounds hearingrelevance tor immunosuppression among the tested only a single alkoxy group in the 8-position. 5-MOPpsoralens. and H37 both show the highest affinity to Kv1.2 I '.

To get same mor~ information on the immuno- followed by Kv1.3 and Kv1.1 (Wulff et al., 1998b). 'Isuppressive potency of the alkoxypsoralens, the ex-. Although Kv1.3 is regarded to be the dominatingpression of a proinflammatory cytokine with major voltage-gated K+ channel type in human and murinepathogenic importance during EAE was investigated. T lymphocytes (Lewis and Cahalan, 1995), rat TFor this investigation, IFN-')' was chosen since it has lymphocytes may express a different K+ channelbeen found to be upregulated during the whole course pattern (Koo et al., 1997). Several K+ channel typesof EAE (McCombe et al., 1998) with the highest may be involved in the regulation of transmembraneexpression levels at the peak of the disease (day 14 potential and intracellular Ca+ in different species.post-immunisation) (Issazadeh et al., 1995). In EAE Therefore, inhibition of immune function may beLNC, we have investigated the IFN-')' mRNA ex- more complete by less specific K+ channel antago-pression by a quantitative RT -PCR with ELISA de- fists than by highly selective K+ channel blockerstection by using a mutagenised internal standard that (Cahalan and Chandy, 1997).allows tor detection of minor differences in the With regard to the therapy of autoimmune de-expression level (down to twofold changes) of 'cy- myelinating diseases, alkoxypsoralens could have atokine genes in cell culture with high accuracy. A second advantage by improving axonal conductivity,significantly stronger suppression of IFN-')' gene ex- which is impaired due to uncovering of normallypression in the LNC through 5-MOP compared to silentjuxtaparanodal K+ channels during the process8-MOP, which was seen in EAE LNC stimulated in of demyelination (Wulff et al., 1998a). In voltage-vitra either specifically with myelin or nonspecifi- clamp experiments on amphibian nodes of Ranvier,

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62 U. Strauss et al./ rmmunophannacology 48 (2000) 51-63 J

5-MOP was found to block delayed rectifier K+ channel genes. In: North, R.A. (Ed.), Handbook of Receptorscurrents (Bohuslavizki et al., 1993). Moreover, sin- and Channels. CRC Press, Boca Raton, pp. 1-71.gle trials have shown that 5-MOP can alleviate func- Ch~mc~nski, P., .Sacchi,. ~.'. 1987: Single-step method of RNA. .. " . . IsolatIon by acid guanldlffium thiocyanate-phenol-chlorofonn

tlonal deficlts In certron MS patients (Bohuslavlzki et extraction. Anal. Biochem. 162, 156-159.al., 1993). Cox, G.W., Orosz, C.G., Fertel, R.H., 1987. 8-Methoxypsoralen

inhibits Iymmphocyte proliferation in vitra in the absence ofultraviolet radiation. Int. I. Immunophannacol. 9, 475-481.

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mltogenesls? Nature 307, 465-468. I"~The authors like to thank MrS. Gabi Köllner foT Gaspar, R., Bene, L., Damjanovich, S., Munoz-Garay, C., ~;

excellent technical assistance, Mrs. Waltraud Ger- Calderon-Aranda, E.S., Possani, L.D., 1995. ß-scorpion toxin 'icke foT valuable help in performing of the animal 2 from Centruroides no.tius blocks voltage gated K+ channelsexperiments and the proliferation assay and Mrs. in human Iymphocytes. Biochem. Biophys. Res. Commun.S L th J' . th . Thi 213, 419-423.usan e am lOT correctlng e manuscnpt. s Hamill, O.P., Marty, A., Neher, E., Sakmann. B., Sigworth, F.I.,work was supported by grants of the Deutsche 1981. Improved patch-clamp techniques tor high-resolution

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genicity while interleukin-2 (1L-2) receptor expression remains

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