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Laboratoire d’Immunologie, Faculté de Médecine, Université de la Méditerranée;
Laboratoire de Neurobiologie des Canaux Ioniques, Unité de Formation et de Recherche de Médecine, Institut National de la Santé et de la Recherche Médicale Unité 464;
Laboratoire de Neurobiologie, Centre National de la Recherche Scientifique, Unité Propre de Recherche 9024;
Laboratoire d’Immunologie, Hôpital Sainte-Marguerite, Institut National de la Santé et de la Recherche Médicale Unité 387; and
¶ Laboratoire d’Ingénierie des Protéines, Institut Federatif de Recherche Jean Roche, Faculté de Médecine-Nord, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6560, Marseille, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, the predominant type of Kv expressed by human, mouse, and
rat T cells is the n type channel, composed of four Kv1.3 
subunits
(12). Kv1.3 is involved in T cell activation and
proliferation (13). High affinity blockers have been used
to demonstrate that Kv1.3 blockade depolarizes the T cell membrane and
attenuates the Ca2+ signaling pathway that is
crucial for lymphocyte activation (14). Because T
lymphocytes must be activated to initiate and support the immune
response, T lymphocyte Kv1.3 represents a valid therapeutic target for
the discovery of anti-inflammatory drugs, as supported by the
suppression of T cell activation by selective peptide and nonpeptide
inhibitors of this channel (toxins, Ref. 15, 16, 17 ;
WIN-17317-3, Ref. 18 ; CP339818, Ref. 19 ;
UK78282, Ref. 20 ; correolide, Ref. 21 ; and
alkoxypsoralens, Ref. 22). This idea has also been
documented by an in vivo mini pig model for delayed-type
hypersensitivity (DTH) (21, 23).
On the basis of these arguments, designing molecules such as KTX that act on neuronal Kv1.1 and T cell Kv1.3 would make it possible to combine the symptomatic treatment of neurological deficits with immunosuppressive effects. To date, none of the selective Kv blockers have been tested for neurological T cell-mediated disease treatment.
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory and
demyelinating autoimmune disease of the CNS, a model widely used to
study MS. EAE is mediated by MHC class II-restricted, myelin
Ag-specific CD4+ T cells (24). These
cells are of the Th1 type, which produce IL-2, IFN-
, and TNF. EAE
can be induced in Lewis rats by adoptive transfer of myelin basic
protein (MBP) T line cells provided these T cells are activated before
injection (25). Because T cell activation is required for
encephalitogenicity, KTX was tested on MBP T cells to block Kv1.3
current, MBP-induced proliferation, activation assessed by cytokine
production and Ca2+ rise, and acquisition of
encephalitogenic properties. Contrary to another study
(26), we established that the immunosuppressive action of
KTX is potent. Also, we found that KTX improves ongoing EAE by at
least 50%.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female inbred Lewis rats 8–10 wk of age and guinea pigs were purchased from Charles River Breeding Laboratories (St. Aubin les Elbeuf, France) and from Iffa Credo (L’Arbresle, France).
Cell lines
The MBP-specific encephalitogenic T cell line, called PAS, had been established from guinea pig MBP-primed Lewis rat lymph nodes. For Ag stimulation, PAS T cells (3 x 105/ml) were incubated for 2–3 days with 10 µg/ml of MBP and 15 x 106/ml syngeneic irradiated (2500 rad) thymocytes as Ag-presenting cells (APC) in RPMI 1640 Dutch modification medium supplemented with 1% syngeneic rat serum and additives. PAS T cells, which produce limited demyelination, had been characterized as cytotoxic, MHC class II-restricted CD4+ T cells that are encephalitogenic in vivo (27, 28). The leukemic human T cell clone Jurkat JH6.2 was a gift from Dr. J. Nunès (U119 Institut National de la Santé et de la Recherche Médicale, Marseilles, France) and maintained as described (29). The murine CTL-L cell line was a gift from Dr. A.M. Schmitt-Verhulst (Centre d’Immunologie, Marseille-Luminy, Marseilles, France) and was grown in RPMI 1640 supplemented with 10% FCS, 50 U/ml human rIL-2, and additives. The murine connective tissue fibroblast cell line L-M was a gift from Dr. A. Fontana (University Hospital, Zurich, Switzerland) and was maintained as described (30).
Reagents
MBP was extracted from guinea pig frozen CNS by the method of
Deibler et al. (31) and purified by C18 reverse-phase HPLC
with a Millipore/Waters Associates system (Milford, DE). The purity was
assessed by electrospray mass spectrometry. KTX was synthesized using
the solid phase method previously described (32).
Synthetic charybdotoxin (ChTX) and -dendrotoxin (-DTX) were
purchased from Latoxan (Rosans, France); synthetic margatoxin (MgTX)
was obtained from Alomone Labs (Jerusalem, Israel).
RT-PCR analysis of Kv1.3 mRNA levels
Total RNAs were extracted from 105 lymphocytes (0, 24, 48, and 72 h after antigenic stimulation), using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. A DNA digestion was performed for 1 h at 37°C with 70 U/ml of RQ1 DNase in 1x reverse transcriptase (RT) buffer (Life Technologies) containing 1000 U/ml of RNase inhibitor (RNasin; Promega, Madison, WI). After heat inactivation of DNase (75°C for 5 min), DNase-treated RNA (500 ng in 10 µl) was reverse-transcribed (33) for 16 h at 37°C by adding 4 µl of 5x RT buffer (Life Technologies), 4 µl of 5x RT mix (containing 2.5 mM deoxynucleotide triphosphate and 24 µM random hexamers), 1 µl RNasin, and 1 µl RT (Superscript II; Life Technologies). Amplifications of cDNA for Kv1.3 and GAPDH were performed by PCR with Hotstar Taq polymerase (Qiagen, Chatsworth, CA), using a Perkin-Elmer thermal cycler. Primer locations (referring to published sequences in GeneBank) were as follows: Kv1.3 (accession number X16001) upper primer 782-802; lower primer 988-968; GAPDH (accession number X02231) upper primer 591-610; lower primer 1042-1023. Conditions of linearity of PCR were determined in preliminary experiments (in terms of amounts of cDNA and of cycle number). After a hot start (94°C for 15 min), cycling conditions were 94°C (45 s), 62°C (1 min), 68°C (1.5 min), with each series of cycles followed by a 4-min elongation step (68°C). Amplification products were analyzed by electrophoresis on 2% agarose gels containing 5 µl/ml ethidium bromide. Images of the gels were numerized (using a Syngene gel imager) and then processed with Molecular Analyst software (Bio-Rad) for quantification of the signal. The ratio of the intensity of Kv1.3 band to the intensity of the GAPDH band was used to determine the relative variations of Kv1.3 transcripts after stimulation. Negative controls included PCR amplification of nonreversed-transcribed RNAs.
Electrophysiological recordings
Whole-cell recordings in PAS and Jurkat JH6.2 T cells were performed according to Mourre et al. (11). KTX was delivered to the bath via a pneumatic pico-pump system (WPI, Aston, U.K.). Dose-response curves were determined by successive additions of KTX at increasing concentrations. Experimental points were fitted to the theoretical hyperbolic curve y = 1/[1 + ([T]/IC50)n], in which y is the fraction of unblocked current, IC50 is the concentration of toxin inducing 50% block, and n is the Hill coefficient. Kinetics of the Kv1.3 blockade induced by KTX was determined as previously described for different toxins (34).
Lymphocyte proliferation assay
The PAS T cells were used after 4–6 days of IL-2-dependent
expansion. They were seeded in flat-bottom 96-well plates (Nunc,
Naperville, IL) in 200 µl of culture medium supplemented with 1%
homologous rat serum, in the presence of APC. K+
channel blockers were added to the cells 30 min before the Ag. The
cells were cultured for 3 days and were pulsed with
[3H]thymidine 16–18 h before being harvested.
The proliferative response of T cells was assessed by a 
scintillation counter measuring the
[3H]thymidine incorporated.
IL-2 and TNF- bioassays
The PAS T cells (3 x 105) and APC
(2 x 106)/0.2 ml were cultured in 96-well
plates. KTX was added 30 min before the Ag. Supernatants (SNs) were
collected 7 and 22 h later and assayed for their biological
activities. The IL-2-dependent CTL-L cell line was used to assay the
IL-2 activity, using mouse rIL-2 as the standard. TNF activity was
determined using TNF--sensitive L-M cells as previously described
(30). TNF- and TNF- activities were distinguished
using a polyclonal TNF--specific rabbit anti-rat Ab (Genzyme,
Cambridge, MA).
[Ca2+]i measurements
The PAS T cells (3 x 106) were
loaded in suspension with 3 µM fura-2 acetomethyl ester (Molecular
Probes, Eugene, OR) for 30 min at 37°C in RPMI 1640 Dutch
modification medium supplemented with 10% FCS. After being washed, T
cells were stimulated by incubating them with IFN--activated and
MBP-pulsed adherent macrophages (3–4 x
106) in the presence or absence of KTX.
Lymphocyte fura-2 fluorescence was measured by a Perkin-Elmer LS-5
fluorescence spectrophotometer with an excitation wavelength at 340 and
380 nm and the emission at 510 nm.
[Ca2+]i was calculated as
previously described (35) using a
fura-2-Ca2+ complex binding constant of 224 nM.
Maximum (Fmax) and minimum
(Fmin) fluorescence values were
obtained by adding ionomycin (1 µM) and then EDTA (20 mM).
Adoptive transfer of EAE and treatment by KTX
One to four million 48-h Ag-activated PAS T cells were injected i.p. into syngeneic rats. The rats were treated by s.c. injections of the indicated doses of KTX in 1 ml of PBS starting the day of the first clinical signs of EAE. Control rats received PBS. The rats were weighed and observed daily. Rats without loss of weight and clinical signs of EAE were considered negative. They were not included in the group of rats from which the mean maximal severity of clinical EAE was calculated.
Measurement of DTH reaction
Ear thickness was measured with a pressure-sensitive micrometer before and 48 h after the intradermal injection of MBP or saline on day 2 after EAE induction.
Statistical analysis
Statistical analysis was conducted using the Mann-Whitney U test. Mean differences between groups were considered significant at values of p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in Fig. 1
, PAS lymphocytes
express significant levels of Kv1.3 mRNA (time 0 after activation). A
signal was readily detectable after amplication of a minute amount of
cDNA (roughly corresponding to 250 pg of total RNA at time 0 after
activation). The amplified fragment for Kv1.3 had the expected size and
comigrated with the amplified fragment from cDNA obtained from rat CNS
(data not shown) or from rat genomic DNA. Levels of Kv1.3 transcripts
were slightly increased (by 40–50%) after a 24-h activation and
plateaued thereafter (48 and 72 h).
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To investigate the in vitro immunosuppressive potential of KTX, we
studied Kv current and activation of PAS T cells in presence of the Kv
blocker. Patch-clamp experiments were performed to document the KTX
effects on currents expressed in PAS. Fig. 1
A (upper
traces) shows the Kv currents recorded in a PAS T cell after 2
days of Ag stimulation. Channels rapidly activated and inactivated upon
depolarization. The voltage for half-activation was -31 ± 2 mV
with a Boltzmann coefficient of 6.7 ± 0.7 mV (n =
5). At 0 mV, the time at which the current reached the peak was
18.1 ± 0.03 ms, and the time constant of inactivation at 0 mV was
258 ± 37 ms. These values correspond to the Kv1.3 characteristics
(36, 37, 38). The KTX effects were tested on Kv1.3 currents
elicited by depolarizing steps 8 min after the application of the
blocker (1 nM, Fig. 1A, lower traces). The
blockade progressively decreased when the KTX perfusion was removed.
However, patch clamping was difficult to maintain on PAS T cells,
probably because of the extreme motility of the membranes, which rarely
makes the seal stable for more than 10 min. Therefore, we further
characterized the properties of the blockade of Kv1.3 induced by KTX in
Jurkat JH6.2 cells, a human T cell leukemia clone constitutively
expressing Kv1.3.
Fig. 1B (left traces) shows that the Kv1.3
currents in Jurkat JH6.2 cells activated and inactivated similarly to
those in the PAS T cells. In Jurkat cells, the voltage required for
half-activation of the conductance was -29 ± 1.3 mV and the
Boltzmann coefficient was 6.1 ± 0.7 mV (n = 9).
At 0 mV, peak time was 10.6 ± 0.09 ms, and the time constant of
inactivation 253 ± 21 ms. The relative blocking potency induced
by KTX was determined by measuring the current remaining at various KTX
concentrations (right traces). Experimental points were
fitted to hyperbolic curves with IC50 = 2 ±
0.07 nM and a Hill coefficient n = 1.02 ± 0.1.
The time constant for blockage (
on) and
recovery (off) was determined using 1, 2.5, 5,
and 10 nM KTX. Fig. 1B shows that
1/on increased linearly with the
KTX concentrations, but 1/off was
constant and independent from the KTX concentrations. Corresponding
rate constants were kon = 2.2 x
106 M-1
s-1 and koff =
8.6 x 10-3 s-1. The
Kd determined by the ratio
koff/kon
was 4 nM, close to that obtained in the precedent dose-response
experiment.
Kv blockers inhibit Ag-induced but not Jurkat JH6.2 T cell proliferation
To determine whether blockers of Kv current would inhibit T cell
proliferation, as was reported for mitogenically stimulated lymphocytes
(15, 39, 40), we performed Ag-dependent T cell
proliferation assays. PAS T cells were cultured with Ag and various
concentrations of three K+ channel blockers. KTX
inhibited the proliferation of the PAS T cells in a dose-dependent
manner with an IC50 of 2.8 nM (Fig. 2A). High inhibition rates
(70–95%) were observed from 6.5 to 100 nM KTX. KTX was nontoxic, as
demonstrated by trypan blue exclusion after a 72-h culture of T cells
with 100 nM KTX. Higher concentrations of the blocker did not further
increase the inhibition (data not shown). Similar results were obtained
with another Kv1.3 blocker, MgTX (data not shown). ChTX, a Kv and
calcium-activated potassium channel blocker, also inhibited the T cell
proliferation (IC50 = 3.8 nM). As expected,
-DTX, a blocker of Kv1.1, Kv1.2, and Kv1.6 (41), all
absent in lymphocytes, had no effect on the PAS T cell
proliferation.
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B). Therefore, K+
blockers inhibit the proliferation of primary T cells, which need an
external stimulus to proliferate, but not of transformed cells. KTX reduces IL-2 and TNF production by MBP-activated T cells
We assayed the IL-2 and TNF contents in culture SNs of T cells
stimulated with MBP and APC in the presence of various concentrations
of KTX. CTL-L and L-M cells were used to measure the IL-2 and TNF
levels, respectively. Control SNs, prepared with APC maintained in
culture for the same period with 100 nM KTX, had no effect in either
biological assay, nor did KTX (100 nM) added directly to the CTL-L and
L-M cell cultures. Fig. 3A
shows that KTX inhibited IL-2 production in a dose-dependent manner.
Complete inhibition of IL-2 production was achieved at 100 nM KTX. In
all the experiments, IC50 was <12.5 nM, a value
similar to those reported by other authors studying the ChTX inhibition
of IL-2 production in anti-CD3- or PHA-stimulated human peripheral
blood T lymphocytes (16, 18, 40). Because IL-2 production
is necessary for PAS T cell proliferation, its addition may restore the
proliferation inhibited in the presence of KTX, as described in the
case of ChTX (15). To test this hypothesis, T cells were
stimulated with MBP and cultured in the presence of IL-2 (0.5, 4, 20,
or 50 U/ml) and KTX at various concentrations (data not shown).
Exogenous IL-2 (4 U/ml) did not increase the T cell proliferation in
the absence of KTX (Fig. 3B). The T cell proliferation was
inhibited by 59% with 100 nM KTX in the presence of IL-2, as compared
with 88% in the absence of IL-2. This partial restoration of the
proliferation was not enhanced by IL-2 in amounts up to 50 U/ml (data
not shown). This finding might be accounted for by the lack of IL-2R
up-regulation that we found when T cells were MBP activated in the
presence of KTX (data not shown). KTX also inhibited TNF production.
TNF levels of SNs without KTX, harvested at 7 and 22 h of culture,
were within the 15- to 25-ng/ml range in two independent experiments.
The biological activity of TNF was consistently reduced by 50% in the
SNs produced in the presence of 100 nM KTX (Fig. 3C).
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One of the earliest events following stimulation is a rise in
[Ca2+]i. To investigate
the effects of KTX on this rise, we measured the
[Ca2+]i of PAS T cells
stimulated by activated macrophages pulsed with MBP. After 10 min of T
cell activation, a measurable, sustained rise in
[Ca2+]i was recorded
(Fig. 3D). This rise was 50% lower in the presence of 300
nM KTX and became equivalent to that recorded in T cells stimulated
with purified protein derivative (PPD), an irrelevant Ag. Hence, KTX
inhibited the specific sustained rise in
[Ca2+]i.
KTX inhibits DTH reaction to MBP
To test the in vivo effects of Kv1.3 blockade, we examined the
ability of KTX to inhibit DTH reaction. Indeed, EAE is associated with
DTH reaction to myelin Ag, here MBP. Rats were injected with
MBP-activated PAS T cells and challenged for DTH reaction 48 h
later; they received MBP intradermally into one ear and PBS into the
other one, as control. DTH response was measured 48 and 72 h
later. KTX was administered twice a day (16 µg s.c.) from the day of
adoptive transfer until the measurements of induration. KTX inhibited
the DTH response by 37–71% (Table I).
Of note, KTX-treated rat CNS displayed no differences in the extent of
cell infiltrations compared with PBS-treated rat CNS except when the
former developed a very mild disease not exceeding a score of 0.5 (data
not shown).
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The concentration of KTX that blocks >80%
[3H]thymidine incorporation (100 nM) found in
preliminary experiments was assayed in vitro to determine whether it
would block the encephalitogenicity. The Kv blocker was present
throughout the 2-day activation culture of PAS T line cells and washed
off along with MBP before inoculation into naive syngeneic rats.
Pretreatment of T cells with KTX clearly attenuated the severity of
adoptively transferred EAE in 15 rats compared with 16 rats injected
with untreated T cells (Table II,
experiments 1 and 3). When KTX was also given in vivo to
Lewis rats during the period of the disease induction, the beneficial
effects became more pronounced, whereas the untreated rats died from
EAE (Table II, experiment 2). Histopathological examinations of CNS of
rats (Table II, experiment 3) indicated that KTX-treated rat CNS
exhibited as many EAE lesions as PBS-treated rat CNS (data not
shown).
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To explore the in vivo efficacy of the Kv blocker for the
treatment of a neurological autoimmune disease, adoptive EAE was
transferred into Lewis rats with MBP-activated PAS T cells. Acute EAE
developed 4–5 days later, lasted for 4–5 days (moderate EAE),
or rapidly evolved to moribund state (lethal EAE). As soon as a
clinical sign was detected, rats were treated with various doses of KTX
for 2 or 3 days. KTX administration reduced the severity of the
disease, prevented its mortality, and shortened its duration (Table III). The beneficial effects of KTX were
dose dependent. In experiment A, when the highest dose (32 µg) was
administered, both the clinical score and duration of EAE were
significantly reduced (p < 0.001 and
p < 0.002, respectively). When 8 µg was given,
differences between clinical severities of the KTX-treated animals
(1.9 ± 1.3) and the control rats (2.7 ± 1.2) were nearly
significant (p = 0.1). The lowest dose (0.8
µg) displayed no effects. In experiment B, in which 8 µg was given,
five KTX-treated rats developed discrete EAE, whereas five control rats
were largely paralyzed and died (p < 0.004).
Comparison of the s.c. and i.v. routes of KTX administration and of the
volumes of KTX in saline injected (0.1 and 1 ml) did not reveal
significant differences (data not shown). Taken together, these results
prompted us to treat animals with the following conditions: 16 µg of
KTX twice a day, in 1 ml PBS, s.c., as early as rats became sick. Of
note, KTX, inhibitor of Kv1.1 and Kv1.3, injected into rats at 32 µg
per day for 3 days, did not cause overt toxicity or neurological
impairment. Pathological studies of CNS confirmed the absence of
effects of KTX (data not shown). Blood biochemistry and
hematology of KTX-treated rats were within the normal range of control
animals (data not shown). In addition, KTX s.c. injected at high doses
of up to 10 mg/kg is reported to induce few detectable symptoms in mice
(32).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that Kv1.3 mRNA transcription in rat encephalitogenic T line cells, specific for MBP, is constitutive and slightly increases by 50% upon activation. This finding is in good agreement with the moderate increases in the peak K+ current density (5- to 10-fold after T cell activation) described in mouse and rat T cells (42, 43). Next, electrophysiological characterization of K+ channel MBP T cells indicated that, with respect to current activation and inactivation, they are similar to the n type K channels in T lymphocytes (43 and 44 , respectively) and to the Kv1.3 expressed in heterologous systems (36, 37, 38). Moreover, current blockade by three peptides known to block Kv1.3 (ChTX, KTX, and MgTX) strongly support the conclusion that the rat T cells used in this study expressed Kv1.3.
We then confirmed that KTX binds to lymphocyte Kv1.3 with an
IC50 of 2 nM. Blockade and unblockade kinetics of
Kv1.3 indicated that one molecule of KTX is sufficient to block one
Kv1.3 (45). The IC50 value
correlates well with the value reported for the KTX blockade of Kv1.3
in human T (26) and B cells (46). Kv and the
calcium-activated potassium channel are thought to be involved in
establishing the T cell membrane potential and providing the electrical
driving force for Ca2+ influx and the subsequent
cell activation (13). Yet, there are conflicting results
on Kv involvement in the activation, probably because of differences
between the T cell activation conditions, the donors, and the
functional T cell assays, and possibly also because of the
participation of other channels in sustaining the requisite membrane
potential (15, 16, 26, 40, 47). In this study, we
eliminated circumstantial variability of this kind by using an
encephalitogenic rat T cell line reactive to MBP to explore several
functions. We found that the IC50 value of KTX
inhibiting T cell proliferation was similar to that found inhibiting
Kv1.3 currents. This similarity suggests that the effects of the toxin
on mitogenesis were mediated by these currents, as proposed for ChTX
(16, 47). Our in vitro results revealed three particular
features. First, the reproducible marked inhibition of the T cell
proliferation observed here contrasts with that described by other
authors (15, 16, 26). In particular, KTX, known to be a
potent selective blocker of the Kv currents in mammalian cells
(11, 26, 37), was suggested to be a weak inhibitor of T
cell proliferation and to slightly reduce steady-state
[Ca2+]i without affecting
peak [Ca2+]i, in
comparison with ChTX (26). That we found KTX to be a
potent inhibitor of T cell proliferation and activation might be
accounted for by differences between experimental protocols but also by
the origin and the concentrations of the serum added to the culture
medium. Note that low serum concentrations favor the antiproliferative
action of Kv blockers (15, 48). Second, we establish that
rat T cells are adequate targets for inhibiting T cell activation by a
Kv1.3 blocker; this is not the case for mouse T cells
(23). Rat MBP T cells have been reported to be good
targets for less specific K+ blockers. Thus, 4-AP
blocks their proliferation and the acquisition of their
encephalitogenicity (49) and the nonpeptidyl blocker,
5,8-diethoxypsoralen (H37, Ref. 22) inhibits whole-cell
K+ currents, their proliferation, and secretion
of IFN- after activation. Thus, one can now test Kv blocker effects
in vivo in well-known experimental rodent models. Third, the KTX
inhibition of the proliferation of MBP-specific T cells was associated
with a marked reduction in TNF production. To our knowledge, this is
the first time the effects of K+ channel blocker
on TNF production have been documented (see Fig. 2C).
It is well known that T cell activation is required for migration into CNS and encephalitogenicity (17, 50, 51). We show that KTX, added to the culture at the beginning of T cell activation, prevented T cells from becoming encephalitogenic. Therefore, it is highly probable that the decrease observed in encephalitogenicity correlated with the drop in Th1 type cytokine production and the inhibition of proliferation caused by the KTX-impairment of Ca2+ signaling. Immunosuppression can be obtained through the regulation of the Ca2+ signaling pathways in T lymphocytes. Yet, to be of interest, the mechanism of action of new compounds should differ from that of the current immunosuppressors such as cyclosporin A. KTX fulfills this requisite. First, KTX does not cross the membrane (52). Next, the mechanism whereby KTX interacts with T cell activation involves a decrease in the Ca2+ flux, which limits the Ca2+-dependent production of IL-2, and ultimately the cell proliferation. Therefore, KTX intervenes earlier in the activation cascade than do cyclosporin A and FK-506. Lastly, because of the restricted tissue distribution of Kv1.1 and Kv1.3, KTX and related blockers might not have such toxic side effects as many of the current immunosuppressive drugs, such as cyclosporin A, FK-506, and rapamycin (14). Overall, these data show that KTX is a potent immunosuppressive agent and that Kv1.3 is a relevant target for immunosuppression.
In vivo, several mechanisms may account for the symptomatic improvement
of EAE by KTX. KTX may exert two kinds of actions: one neurological,
the other immunosuppressive. We provide evidence that KTX exerted
immune suppression in vivo because KTX treatment decreased DTH response
in Lewis rats. KTX effects could come into play at the level of
migration of T cells from the blood vessels into tissues including CNS.
However, we did not find evidence of major differences between the
inflammatory lesions of CNS of rats injected with KTX-treated T cells
and untreated T cells, and between the levels of adhesion molecule
expression, in particular, very late antigen (VLA)-4, of T cells
activated in the presence and in the absence of KTX (data not shown),
Hence, our results are in agreement with neither this hypothesis nor
with the results of Levite et al. (53). They demonstrate
that elevated levels of
(K+)0-induced T cell
adhesion to fibronectin-coated wells is inhibited by specific Kv1.3
blockers and that Kv1.3 is physically and functionally linked to
integrin moieties. Alternatively, the fact that KTX interfered with T
cell functions, resulting in the peripheral immunosuppression observed,
could be due to an inhibition of de novo Ag activation. We further show
that KTX down-regulated the production of IL-2 and TNF by
encephalitogenic T cells. TNF- and TNF- play a role in the
inflammation and in the formation of demyelinating lesions in EAE
(54). Because blocking TNF- prevents EAE (55, 56), both of these KTX-induced effects would interfere in vivo
with the development of EAE. KTX could also inhibit the production of
other proinflammatory cytokines and/or enhance that of
contra-inflammatory ones such as TGF-, which is increased by
cyclosporin A in human T cells (57). Interestingly,
TGF- mediates suppression in EAE (58). On the basis of
these data, KTX may play an important role on the immune system in
inhibiting EAE.
Besides, KTX may exert an action on the CNS during EAE. No single animal model mimics all the features of human demyelinating diseases; rather, the available models reflect specific facets. Here, we focus on acute EAE in Lewis rats. Acute EAE can be considered to roughly mimic the initial attack of MS (59). Besides, adoptive EAE was chosen because highly pathogenic T cell lines can be easily established from this species and this strain. In that model, clinical signs run in a more synchronous course, which makes the results of drug treatment easier to analyze. Moreover, note that the encephalitogenic T cell line used here produces demyelination, although limited. Demyelination resulting from adoptive transfer of MBP T cells has already been reported (60, 61, 62 ). Paralysis has been shown to parallel the slowing of conduction in the CNS (61). A broad spectrum Kv blocker such as 4-AP restores conduction to fibers demyelinated in vitro (4, 5, 6, 7) and potentiates synaptic transmission (7, 8), effects which could account for the beneficial action of 4-AP in MS patients. Yet, 4-AP could also affect the immune system of MS patients because it can block T cell activation (18, 63). However, supraclinical concentrations (mM range) of 4-AP are necessary to obtain this immunosuppression, which makes the action of 4-AP on the immune system unlikely. Moreover, the prompt onset of at least some of the beneficial effects of 4-AP therapy in patients supports the notion of direct effects on the CNS, whatever the mechanism may be. Note that in contrast to 4-AP, immunosuppression is obtained by similar KTX concentrations in vivo and in vitro (10–50 nM).
In conclusion, we confirm that Kv1.3 is a relevant target for immunosuppression and we demonstrate that KTX is a potent candidate drug for treating autoimmune pathologies. Moreover, because KTX can also block Kv1.1, it may play a role directly at the neurological level, a role that could be reflected in its beneficial symptomatic effects in EAE. This role has to be investigated. Our studies point the way to future work to develop selective Kv1.3 and Kv1.1 blockers for treating autoimmune-demyelinating neurological diseases.
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Acknowledgments |
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2 Address correspondence and reprint requests to Dr. Evelyne Beraud, Laboratoire d’Immunologie, Faculté de Médecine, Université de la Méditerranée, 27 Bd Jean Moulin, 13385 Marseille cedex 5, France.
3 Abbreviations used in this paper: Kv, voltage-gated potassium channel(s); EAE, experimental autoimmune encephalomyelitis; MS, multiple sclerosis; MBP, myelin basic protein; KTX, kaliotoxin; ChTX, charybdotoxin; MgTX, margatoxin; -DTX, - dendrotoxin; DTH, delayed-type hypersensitivity; AP, aminopyridine; SN, supernatant; RT, reverse transcriptase.
Received for publication June 12, 2000. Accepted for publication October 24, 2000.
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), ChTX (
), or 
) rIL-2 (4 U/ml) added at the same time (