© 1996 American Heart Association, Inc.
Articles |
Interaction of Cytotoxic T Lymphocytes and Guinea Pig Ventricular Myocytes
Pharmacological Modulation by Blocking K+ Currents in Cytotoxic T Lymphocytes
From the Rappaport Family Institute for Research in the Medical Sciences (B.F., R.L., O.B.), Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel; Merck Research Laboratories (M.G.), Rahway, NJ; the Department of Cell Biology (G.B.), Weizmann Institute of Science, Rehovot, Israel; and the Department of Molecular Pharmacology (P.G.), Stanford (Calif) University School of Medicine.
Correspondence to Ofer Binah, DSc, Rappaport Institute, PO Box 9697, Haifa 31096, Israel.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and Methods Results Discussion References |
|---|
Abstract Infiltrating cytotoxic T lymphocytes (CTLs) are important immune effectors that damage the myocardium during heart transplant rejection as well as in cardiomyopathy and Chagas' heart disease. We have previously shown that in an in vitro model of murine-derived peritoneal exudate CTL (PEL)–guinea pig ventricular myocyte interaction, PEL induced in conjugated myocytes reduction of resting membrane potential and action potential (AP) amplitude, shortening of AP duration, delayed afterdepolarizations (DADs), and myocyte contracture and destruction. Since these findings indicated that cytotoxicity was largely caused by [Ca2+]i overload, in the present study we tested the hypothesis that blocking the L-type Ca2+ current (ICa,L) in the myocyte will eliminate the trigger for Ca2+ release from intracellular stores and will reduce [Ca2+]i overload and subsequent myocyte deterioration. CoCl2 (3 mmol/L) prevented PEL-induced AP changes, induction of DADs, and myocyte destruction. Since verapamil (2 µmol/L) was ineffective, indicating that the CoCl2 protection was not due to block of ICa,L, we tested whether the different action of these Ca2+ channel blockers was due to their differential effect on the PEL's K+ current (IK), previously shown to participate in lymphocyte activation and cytotoxicity. In agreement with their protective efficacy, CoCl2 but not verapamil blocked IK in PELs, suggesting that this is the mechanism for the protection provided by CoCl2. To support this notion, we tested the effect of the scorpion-derived peptide margatoxin (10 nmol/L), a specific K+ channel blocker in lymphocytes, on PEL-myocyte interaction and on PEL's IK; margatoxin prevented PEL-induced cytotoxicity and also blocked IK in PEL. Based on these findings, an alternative modality for attenuating CTL-induced lymphocytotoxicity is proposed.
Key Words: cytotoxic T lymphocytes • peritoneal exudate cytotoxic C lymphocytes • ventricular myocytes • heart transplant rejection • Ca2+ channel blockers
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Cardiac transplantation is now an accepted treatment for end-stage myocardial failure, with the major obstacle to higher survival rates being the immunological rejection of the transplanted heart. Once an allograft is recognized as nonself, rejection occurs by means of two pathways, cellular and humoral, which operate in concert to destroy foreign tissues. The cellular reaction is initiated mostly by helper T (CD4+) lymphocytes, which were initially sensitized by the graft antigens (mainly major histocompatibility complex class II) brought to them by antigen-presenting cells and by the donor endothelium. Once sensitized, helper T lymphocytes trigger the production of allograft-specific CTLs, which then destroy the nonself tissues. At the same time, helper T lymphocytes recruit macrophages into the rejection site by releasing various cytokines. In turn, macrophages release interleukin-1, which further stimulates the system by promoting interleukin-2 production by T lymphocytes. The allospecific CTLs then attack alloantigens, mainly those belonging to the class I major histocompatibility complex antigens. Consequently, the cytolytic machinery is activated, and the CTLs stage a lethal attack to destroy donor cells displaying nonself antigens.1 2
Lymphocyte-induced damage to the cardiac muscle is not restricted to heart transplant rejection but is evident in other heart diseases. It is commonly thought that myocarditis, initiated by a viral infection, often coxsackievirus B3, may progress in a significant number of cases to dilated cardiomyopathy. Although the precise pathological mechanisms of dilated cardiomyopathy are far from clear, there is a rather wide agreement that CTL-mediated immune response contributes to the ongoing cardiac damage, often leading to terminal heart failure.3 4 5 In addition, CTLs have been implicated in myocardial damage induced in Chagas' heart disease in humans and also in the chronic model of Chagas' heart disease in Trypanosoma cruzi–infected mice.6 7
To understand the precise nature of the interaction of infiltrating lymphocytes and myocytes during heart transplant rejection as well as during the course of other heart diseases associated with infiltrating CTLs, in a recent study we established an in vitro model of interaction of CTLs and guinea pig ventricular myocytes.8 We used patch-clamp techniques to monitor action potentials and membrane currents in single ventricular myocytes undergoing cytocidal interaction with killer lymphocytes. We showed that CTLs caused general decline in action potential characteristics and in myocyte contracture; these effects can account, at least in part, for the decline in cardiac function during heart transplant rejection and in related heart diseases. On the basis of these findings, we proposed that cytotoxicity was essentially caused by [Ca2+]i overload in conjugated myocytes.
In spite of inherent limitations of an isolated CTL-myocyte conjugate as a model of CTL-mediated heart diseases, it is currently the only experimental model enabling electrophysiological recordings from an "identified" CTL-myocyte conjugate undergoing cytocidal interaction.8 Therefore, we thought that this model was a valuable tool for investigating drugs that may interfere with the cytocidal interaction either at the killer and/or at the target cell site. Because we have previously suggested that PEL-induced damage is associated with [Ca2+]i overload in the myocyte, in the present study we investigated the effects of Ca2+ channel blockers. We hypothesized that blocking ICa,L by means of common Ca2+ channel blockers, such as CoCl2 and verapamil, will eliminate the main trigger for Ca2+ release from intracellular stores, mainly the sarcoplasmic reticulum, and will therefore reduce [Ca2+]i overload and myocyte deterioration.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Preparation of Ventricular Myocytes
Adult guinea pig ventricular myocytes were obtained by an enzymatic dissociation procedure.9 Guinea pigs (350 to 400 g) were anesthetized intraperitoneally with sodium pentobarbital (30 mg/kg). The chest was opened, and the heart was rapidly removed, cannulated through the aorta, and perfused with modified Tyrode's solution in a Langendorff apparatus. After washing out the blood, the heart was perfused with 50 mL of nominally Ca2+-free Tyrode's solution. Subsequently, Ca2+-free Tyrode's solution containing 0.04% to 0.05% collagenase (Worthington Biochemical Corporation) was circulated for 25 to 35 minutes. Thereafter, the heart was perfused with 50 mL of KB medium.9 All solutions were oxygenated and warmed to 36.0±0.2°C. Finally, the heart was removed from the cannula and placed in KB medium, and then the suspension was filtered through a nylon mesh and stored in KB medium at room temperature (24.0°C to 25.0°C) before the experiment. The modified Tyrode's solution contained (mmol/L) NaCl 140, KCl 4, CaCl2 1.8, MgCl2 1, glucose 10, and HEPES 5. The KB medium contained (mmol/L) KCl 70, K2HPO4 30, MgSO4 5, CaCl2 0.12, glucose 20, taurine 20, succinic acid 5, pyruvic acid 5, creatine 5, Na2ATP 5, and EGTA 1.
Electrophysiological
Recordings
After 1 to 2 hours of incubation in KB medium, myocytes
were
transferred to the recording chamber (0.5 mL), which was
mounted on the stage of the inverted microscope (Zeiss IM). The bath
was superfused with Tyrode's solution at a rate of 1 to 2 mL/min, and
all experiments were carried out at room temperature (24.0°C to
25.0°C). Membrane potentials and currents were measured by means of
the whole-cell recording technique10 with an
EPC-7 (List Medical Electronics) or Axon 200A (Axon Instruments, Inc)
patch-clamp amplifier. Electrodes were prepared from glass
micropipettes (H15/10, Jencons Scientific), pulled on a vertical puller
(Narashige PP-83), and had a tip resistance of 2 to 4 M
when filled
with the pipette solution containing (mmol/L) potassium aspartate 120,
KCl 20, MgCl2 3.5, KH2PO4 20,
Na2ATP 3, glucose 10, and EGTA 1.
PELs
In vivo primed PELs were obtained from BALB/c mice 4 to
5 days
after the secondary intraperitoneal immunization
with 25x106 C57BL leukemia EL4
cells.11 At the day of the experiment, mice were killed by
cervical dislocation, and peritoneal exudates were obtained by rinsing
the peritoneal cavities with PBS containing 10%
heat-inactivated NCS. The peritoneal exudate cells were
centrifuged, resuspended in RPMI+NCS+HEPES (10 mmol/L), and
incubated in Petri dishes at 37.0°C to cause adherence of B cells and
macrophages to the plastic. After 60 minutes, the nonadherent
cells (PELs) were centrifuged again, then eluted by
RPMI+NSC+HEPES, and kept on ice. The cellular composition and
surface
markers (analyzed by FACS) of PELs are as follows: small to
medium-sized lymphocytes (91%), histiocytes (<2%), and
polymorphonuclear cells (<2%); Thy 1.2 (95%), Lyt-2 (82%), and
L3T4 (19%).12 13 All procedures for handling animals
were
in accordance with institutional guidelines.
The Experimental Model: Conjugate Formation Between Myocytes
and CTLs
CTL-myocyte conjugates were formed between mouse BALB/c
anti-EL4
CTLs (PELs) and guinea pig ventricular myocytes pretreated
with the plant lectin Con A before exposure to PELs. The
sugar-binding lectin "bridges" over the specific recognition
step between the killer and the xenogeneic (guinea pig)
myocytes.14 After obtaining control measurements from a
nonconjugated myocyte, flow to the bath was stopped, and Con A was
added to a final concentration of 10 µg/mL. Ten minutes later, a drop
(10 to 20 µL) of PEL suspension was added to the bath, resulting in
myocytes to which several PELs were attached (see Fig 1A
). That
PELs
and myocytes were firmly bound (ie, conjugated) was verified by gently
moving the recording pipette in the bath; in the presence of
Con A, PELs strongly adhered to the "cruising" myocyte; in its
absence, the lymphocyte detached.
|
Statistical Analysis
Results are expressed as
mean±SEM. To compare means of two
populations, we used Student's t test for paired or
unpaired observations.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Morphological and Electrophysiological Alterations in Myocytes Conjugated With PELs: Effects of CoCl2 and Verapamil
The morphological and electrophysiological alterations in myocytes conjugated with PELs have been reported previously,8 with the major morphological change being progressive shortening, culminating in hypercontracture and destruction (Fig 1A
). We evaluated these changes by two
morphological markers: (1) the first indication of myocyte shortening,
28.9±2.8 minutes after formation of conjugates (n=13), and (2)
myocyte
destruction, 43.5±4.3 minutes after conjugate formation. Such
morphological deterioration may reflect CTL-induced myocardial damage.
Fig 1B depicts typical changes in the action potential of a
conjugated
myocyte. While the action potential was stable for the entire duration
of the experiment (60 minutes) in a nonconjugated Con A–treated
myocyte (or in myocytes exposed to PELs in the absence of Con A [not
shown]), the action potential was severely altered in conjugated
myocytes, as indicated by reductions in Vm and APA, a
marked shortening of APD, and attenuation of the plateau. Frequently,
these alterations were associated with delayed afterdepolarizations
(shown by the arrows), indicative of
[Ca2+]i
overload. PEL-induced changes in action potential characteristics are
summarized in the Table.
|
To test the hypothesis that
blocking ICa,L, which
triggers Ca2+ release from the sarcoplasmic reticulum, will
reduce [Ca2+]i overload and the
subsequent
cytocidal damage, we determined whether the potent Ca2+
channel blocker CoCl2, recently found to attenuate
the lytic effects of CTL lytic granules on ventricular
myocytes,15 can modify PEL-myocyte cytocidal interaction.
In control experiments, we determined the effect of CoCl2
(3 mmol/L) on action potential characteristics of nonconjugated
myocytes (Fig 2A, upper trace) and found, as expected,
that CoCl2 attenuated the plateau and shortened APD. As
shown in the Table, whereas Vm and APA were
unchanged
during 60 minutes of superfusion with CoCl2,
APD50 was reduced. To test the ability of CoCl2
to modulate PEL-myocyte cytocidal interaction, the drug was introduced
to the superfusate immediately after the addition of PELs
and conjugate formation and remained in the bath for the entire
duration of the experiment. This procedure was used to prevent a
possible effect of CoCl2 on conjugate formation. As seen by
the representative recordings in Fig 2A (lower
trace), the action potential of the CoCl2-treated
conjugated myocyte is indistinguishable from that of
CoCl2-treated nonconjugated myocyte, indicating that
CoCl2 provided considerable protection to the conjugated
myocyte. Aside from the expected APD shortening by CoCl2 in
the conjugated myocyte (as in the nonconjugated myocyte),
Vm and APA were not reduced, the plateau was not abolished
(compare with Fig 1B) (summarized in the Table),
and most important,
delayed afterdepolarizations were not induced. In agreement with these
findings, PEL-induced myocyte contracture and destruction did not occur
in CoCl2-treated conjugated myocytes.
|
To determine whether
the protective potency of CoCl2 was
due to a block of ICa,L, we tested whether
verapamil (2 µmol/L), another Ca2+ channel
blocker, had a comparable protective effect. Unlike
CoCl2, verapamil failed to protect
myocytes against killer lymphocytes (Fig 2B and
Table), and action
potential deterioration occurred as under drug-free conditions.
Accordingly, verapamil did not prevent the induction of
delayed afterdepolarizations (Fig 2B, lower trace) or myocyte
contracture and destruction, indicating that ICa,L was not
directly involved in the damage induced in myocytes by killer
lymphocytes.
Changes in IK During PEL-Myocyte Interaction: Effect of
CoCl2
Marked APD shortening and attenuation of the plateau
are the major
electrophysiological alterations in
conjugated myocytes. Hence, we addressed two related questions: (1)
Does an increase in IK (affecting the plateau and
repolarization of the action potential) in conjugated myocytes
contribute to the above changes in action potential? (2) Did
CoCl2 prevent the changes in action potentials of
conjugated myocytes because of its effect on IK per se? In
other words, can a direct inhibitory effect of
CoCl2 on IK potentially oppose a PEL-induced
([Ca2+]i-dependent) increase in
IK? Theoretically, it can be argued that experiments
testing the effect of CoCl2 on IK could have
been more conclusive if performed with conjugated myocytes rather than
with nonconjugated myocytes; however, we chose the second option, since
the possibility of an "indirect" effect of CoCl2 on
IK (in myocytes), mediated through the drug's effect on
the lymphocyte, could not be excluded. Fig 3A depicts
representative membrane currents in a nonconjugated
myocyte at 0 and 60 minutes (after the beginning of the experiment);
these experiments served as the control for testing the changes in
IK in conjugated myocytes. To prevent a possible effect on
the cytocidal process (in the myocyte and/or the lymphocyte), we did
not use ion channel blockers (eg, tetrodotoxin) or ion-substitution
protocols in order to obtain "pure" IK. The decline
in IK with time seen in this experiment probably resulted
from the natural current "run-down," commonly occurring with
other currents (eg, ICa,L). A summary of the decline in
IK over time (0 to 60 minutes) in nonconjugated myocytes is
depicted by IK I-V relations (Fig 3B). In contrast
to the
moderate decline seen in control experiments, in conjugated myocytes
(Fig 3B), the interaction with PELs resulted in a marked
increase in
IK, which can account, at least in part, for APD
shortening and for the plateau attenuation. The mechanism responsible
for the increase in IK in conjugated myocytes was not
explored directly in the present study. We next addressed the
second question, testing whether CoCl2 affects
IK in nonconjugated myocytes. As seen by the
representative membrane currents (Fig 4A) and by the I-V
relations (Fig 4B),
CoCl2 only minimally affected IK.
|
|
Effects of Verapamil and CoCl2 on
IK in PELs
The observation that two potent
Ca2+ channel blockers,
CoCl2 and verapamil, differed markedly in their
ability to interfere with PEL-induced cytotoxicity implied that a block
of ICa,L in conjugated myocytes is unlikely to provide
protection against the cytotoxic damage. This observation, along with
the finding that IK, which is markedly increased in
conjugated myocytes, is also not a target for CoCl2,
suggested that the protection provided by CoCl2 may have
been due to the drug's effect on membrane currents of the killer
lymphocyte. Ideally, testing the effect of CoCl2 on PEL
currents should have been performed on active lymphocytes conjugated to
a myocyte; however, since in every experiment, several lymphocytes
adhered to a myocyte (Fig 1A), it could not be determined
whether
a randomly monitored PEL is indeed the one killing the myocyte.
Furthermore, it is extremely difficult (if not impossible) to determine
how the presence of a patch electrode on the lymphocyte and dilution of
the lymphocyte intracellular content affect the cytotoxic process. To
bypass this uncertainty, experiments were performed on nonconjugated
PELs. Typical PEL whole-cell membrane currents are depicted in Fig
5A. The expansion of the first 5 milliseconds (Fig
5B)
demonstrates that the membrane current closely resembles common
IK, with a relatively rapid onset and
voltage-dependent kinetics. The I-V relations of current measured
at 22 milliseconds are depicted by the open circles in Fig 6.
|
|
) and 10 minutes after superfusion with the drug (
). A,
The holding potential was -50 mV, and the voltage-clamp
pulses ranged from -30 to +60 mV at 10-mV intervals. B, The
holding potential was -50 mV, and the voltage-clamp pulses
ranged from -80 to +60 mV at 10-mV intervals. In both panels,
interpulse intervals were 20 seconds.
Since a variety of membrane currents, and specifically
K+
currents, have been associated with lymphocyte activation and
proliferation,16 17 18 it was of
importance to determine
whether the ability of CoCl2 and the inability of
verapamil to protect myocytes coincide with their effects
on PEL membrane currents. As seen by the effect of both drugs on the
I-V relations (Fig 6), whereas verapamil was practically
ineffective, CoCl2 almost completely blocked IK
in PELs, providing a plausible explanation for the drug's ability to
protect myocytes against PEL-induced cytotoxic damage.
Margatoxin Protects Conjugated Myocytes and Blocks IK
in PELs
Because the above experiments suggested that blocking
IK in PELs prevented damage induced by the lymphocytes, we
used margatoxin, a specific blocker of IK in T lymphocytes.
Margatoxin, a peptide isolated from the venom of the scorpion
Centruroides margaritatus, has been shown to block cloned
rat Kv1.3 channels19 20 as well as
IK in PEL hybridomas (O. Binah, P. Gardner, unpublished
data, 1995). First, we tested whether margatoxin has a protective
effect comparable to that of CoCl2. As seen in Fig 7
(and summarized in the Table), the presence of 10
nmol/L margatoxin (a concentration previously shown to block
IK) prevented the typical PEL-induced deterioration in
action potential characteristics and the induction of delayed
afterdepolarizations. Margatoxin also prevented myocyte contracture. To
exclude an effect of margatoxin (at 10 nmol/L) on myocytes that might
be related to its protective efficacy, we determined whether margatoxin
affects the action potential or membrane current of nonconjugated
myocytes. At the same concentration that margatoxin blocked PEL-induced
damage, it did not affect action potential characteristics
(Table) or
IK (Fig 8). Finally, we determined whether
the protective capacity of margatoxin corresponds with its ability to
block IK in PEL (Fig 9). As anticipated,
margatoxin blocked IK almost completely in PELs, leaving a
small current component, most likely due to a "leakage current."
Therefore, this observation strongly supports the findings with
CoCl2 (and verapamil) suggesting that blocking
IK in PELs prevents delivery of the cytotoxic message (ie,
the "lethal hit") to the myocyte.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
The experimental model of CTL-myocyte interaction was developed in order to investigate the cellular mechanism(s) responsible for myocardial damage induced by infiltrating lymphocytes during the immunological rejection of transplanted hearts. This model is also relevant to other heart diseases associated with CTL infiltration, such as viral myocarditis and Chagas' disease. Since attenuating CTL cytotoxicity (ie, "immunosuppression") is a major goal in retarding lymphocyte-mediated immunopathology, our overall objective was to determine whether the damage induced by CTL to myocytes can be modulated by pharmacological modification of membrane currents in conjugated myocytes and/or in CTL. Heart transplant rejection is a complex multifactorial process that cannot be duplicated reproducibly in vitro by an isolated CTL-myocyte conjugate. Nonetheless, it is presently a unique (and only) experimental model enabling whole-cell recordings from a single conjugated myocyte undergoing cytocidal interaction with a killer lymphocyte. Regarding the fact that we have studied cellular (xenogeneic) interaction between BALB/c anti-EL4 PELs and Con A–treated guinea pig ventricular myocytes, to the best of our knowledge, all evidence supports the view that CTLs use similar mechanisms to lyse allogeneic and xenogeneic target cells, either mediated by lectin (eg, Con A) or not.11 Additionally, with the growing demand for clinical xenografts, the present system offers important information in a field of increasing interest. The major findings were as follows: (1) CoCl2 and margatoxin, but not verapamil, prevented PEL-induced changes in action potential and myocyte destruction. (2) Since CoCl2 and verapamil, both potent ICa,L blockers, differed in their protective potency, this protection was not due to a block of ICa,L in ventricular myocytes. (3) None of the drugs tested affected IK in myocytes. (4) In agreement with their protective efficacy, CoCl2 and margatoxin, but not verapamil, blocked IK in the killer lymphocyte, suggesting that this is the mechanism for the protection provided by CoCl2 and margatoxin. Based on these findings, an alternative modality for attenuating cellular cytotoxicity, namely, immunosuppression, should be examined in future studies.
We have recently reported that PEL-myocyte interaction results in a reduction in Vm, APA, and APD, as well as contracture and cell destruction. We concluded that some effects may be accounted for by [Ca2+]i overload. We suggested that the marked APD shortening could result from (1) [Ca2+]i-dependent increase in the repolarizing outward current, IK,21 and (2) decreased ICa,L resulting from Ca2+-dependent inactivation.22 In the present study, we demonstrated that PEL-myocyte interaction was associated with a marked increase in IK, probably caused by increased [Ca2+]i. The possibility that APD shortening was due to Ca2+-dependent ICa,L inactivation is less likely, since shortening occurred in the presence of verapamil and in the notable absence of ICa,L.
Our original working hypothesis was that blocking ICa,L will attenuate PEL-induced damage to conjugated myocytes as a result of decreased [Ca2+]i overload. That this assumption was incorrect was suggested by the findings that two potent Ca2+ channel blockers differed in their ability to protect myocytes; although verapamil was ineffective, CoCl2 provided significant protection. We then concluded that the conjugated myocyte was not the target for the protective action of CoCl2 and directed our experiments to the lymphocyte. Under normal experimental conditions, we recorded one type of membrane current in PEL, which is characterized by rapid-onset voltage-dependent kinetics and the absence of inactivation (even at pulses of 200-millisecond duration [not shown]). We found that in complete agreement with the drug's ability (or inability) to protect conjugated myocytes, PEL membrane current, carried mainly by IK, was blocked by CoCl2 but not by verapamil. Although we have not carried out a comparative analysis of the current characteristics, of the three known types of IK in lymphocytes, the current sensitivity to CoCl2 and margatoxin and insensitivity to verapamil suggest that it is the l-type IK.16 17 18 23
Having found that blocking IK in PELs prevented damage to
conjugated myocytes, we tested the protective efficacy of margatoxin, a
specific K+ channel blocker in
lymphocytes.24 25 26 As anticipated from
previous reports,
margatoxin blocked IK in PELs and provided complete
protection to conjugated myocytes. At the same time, margatoxin did not
affect the action potential or IK of nonconjugated
myocytes, suggesting that the myocyte was not the target for the
protective action of margatoxin. Margatoxin is a 39–amino-acid
peptide toxin recently isolated from the New World scorpion
Centruroides margaritatus, and its primary structure has
been determined.26 27 Margatoxin is structurally
related
to other well-known scorpion toxins and displays 44% sequence
identity with charybdotoxin, 41% with iberiotoxin, and 79% with
noxiustoxin. Previous studies have shown that margatoxin blocks
voltage-dependent Kv1.3 current in human peripheral T
lymphocytes with high affinity (half block at 
50
pmol/L)27 and has no effect on the small-conductance
Ca2+-activated K+ channels in human T
lymphocytes.24 In smooth muscle sarcolemma, margatoxin,
unlike charybdotoxin, is inactive on the high-conductance
Ca2+-activated K+ channel even at 1
µmol/L.27
Why Does Blocking IK in Killer Lymphocytes Prevent the
Cytotoxic Damage to Conjugated Myocytes?
The involvement of different
ion channels in T-lymphocyte
activation and proliferation is well
established,16 17 18 but
less is known about the role of ion channels in cytotoxic function.
Several observations suggest that K+ currents are involved
in cytotoxicity. 86Rb efflux from CTLs was increased under
conditions of specific target cell lysis.28 Pretreatment
of natural killer cells with quinidine,
4-aminopyridine, verapamil, and
CdCl2, at concentrations that block K+
channels, inhibited the killing of target cells.29
Similarly, the K+ channel blockers quinidine and
4-aminopyridine inhibited target cell lysis by
lymphokine-activated killer cells.30 Strong
support for the involvement of K+ channels in lymphocyte
activation and cytotoxic function comes from a recent study testing the
effects of margatoxin in human T lymphocytes.24 At
concentrations at which margatoxin blocked voltage-dependent
K+ channels in lymphocytes (0.5 to 5 nmol/L), the toxin
inhibited interleukin-2 production of ionomycin+phorbol
12-myristate 13-acetate–stimulated T lymphocytes, as well
as the anti-CD3–induced rise in
[Ca2+]i.
These observations strongly support the findings of the present
work, ie, that blocking IK prevented killer lymphocyte
activation and delivery of the lethal hit to the target cell. Finally,
it should be noted that at present the precise role of
K+ channels in lymphocyte activation and cytotoxic function
is not entirely clear. Since depolarizing the cell with high external
K+ enhanced killing whereas hyperpolarizing the cell with
valinomycin or low external K+ inhibited killing, the
primary role of K+ channels is probably not only to
maintain a negative resting potential.31 Thus, although
there is a correlation between K+ channel blockade and
inhibition of lymphocytotoxicity, the mechanism(s) whereby
K+ channels are involved in killing is yet to be
determined.18
In summary, we investigated the cytocidal interaction of PELs and ventricular myocytes and demonstrated that blocking IK in PELs by CoCl2 and margatoxin prevented PEL-induced damage to the myocytes. This pharmacological modulation of lymphocytotoxicity in vitro can be considered a potential modality for a novel immunosuppression therapy.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by grants to Drs Binah and Berke from the US-Israel Binational Science Foundation, the Israeli Ministry of Health, the Minerva Foundation through the Bernard Katz Center for Cell Biophysics, and the German Cancer Center, Heidelberg. The research was also supported by the Rappaport Family Institute for Research in the Medical Sciences and by a grant to Dr Gardner from the American Cancer Society (DB26E).
Received May 5, 1995; accepted October 31, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
1. Auchincloss HD, Sachs H. Transplantation and graft rejection. In: Paul WE, ed. Fundamental Immunology. New York, NY: Raven Press Publishers; 1989:889-922.
2.
Binah O. Immune effector mechanisms in heart
transplant rejection. Cardiovasc Res. 1994;28:1748-1757.
3.
Leslie K, Blay R, Haisch C, Lodge A, Weller A, Huber
S. Clinical and experimental aspects of viral
myocarditis. Clin Microbiol Rev. 1989;2:191-203.
4.
Seko Y, Shinkai Y, Kawasaki A, Yagita H, Okumura K,
Takaku F, Yazaki Y. Expression of perforin in infiltrating cells
in murine hearts with acute myocarditis caused by coxsackievirus
B3. Circulation. 1991;84:788-795.
5.
Huber SA, Morasaka A, Choate M. T cells
expressing the 
T-cell receptor potentiate coxsackievirus
B3-induced myocarditis. J Virol. 1992;66:6541-6546.
6. Brener Z. Pathogenesis and immunopathology of chronic Chagas' disease. Mem Inst Oswaldo Cruz. 1987;82:205-213.
7.
Morris SA, Tanowitz HB, Wittner M, Bilezikian
JP. Pathophysiological insights into the
cardiomyopathy of Chagas' disease.
Circulation. 1990;82:1900-1909.
8. Felzen B, Berke G, Rosen D, Binah O. Mechanisms whereby cytotoxic T lymphocytes damage guinea pig ventricular myocytes. Pflugers Arch. 1994;427:422-431. [Medline] [Order article via Infotrieve]
9. Isenberg G, Klockner U. Calcium tolerant ventricular myocytes prepared by preincubation in a `KB medium.' Pflugers Arch. 1982;395:6-18. [Medline] [Order article via Infotrieve]
10. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high resolution recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85-100.[Medline] [Order article via Infotrieve]
11. Berke G, Sullivan KA, Amos DB. Rejection of ascites tumor allografts, l: isolation, characterization and in vitro reactivity of peritoneal lymphoid effector cells from BALB/c mice immune to EL4 leukosis. J Exp Med. 1972;135:1334-1350. [Abstract]
12. Berke G. Interaction of cytotoxic T lymphocytes and target cells. Prog Allergy. 1980;27:68-133.
13. Berke G, Rosen D. Highly lytic in vivo primed CTL devoid of lytic granules and BLT-esterase activity acquire these constituents in the presence of growth factors upon blast transformation in vivo. J Immunol. 1988;141:1429-1436. [Abstract]
14.
Sharon N, Lis A. Lectins as cell recognition
molecules. Science. 1989;246:227-233.
15.
Binah O, Berke G, Rosen D, Hoffman B. Calcium
channel blockers modify
electrophysiological effects of lytic
granules from cytotoxic T lymphocytes in guinea pig
ventricular myocytes. J Pharmacol
Exp Ther. 1994;268:1581-1587.
16. Gallin EK. Ion channels in leukocytes. Pharmacol Rev. 1991;71:775-811.
17. Lewis RS, Cahalan MD. Ion channels and signal transduction in lymphocytes. Annu Rev Physiol. 1990;52:415-430. [Medline] [Order article via Infotrieve]
18. Premack BA, Gardner P. Role of ion channels in lymphocytes. J Clin Immunol. 1991;11:225-238. [Medline] [Order article via Infotrieve]
19.
Grissmer S, Dethlefs B, Wasmuth JJ, Goldin AL, Gutman
G, Cahalan MD, Chandy KG. Expression and chromosomal
localization of a lymphocyte channel gene. Proc Natl Acad
Sci U S A. 1990;87:9411-9415.
20. Novick J, Leonard RJ, King VF, Schmalhofer W, Kaczorowski GJ, Garcia ML. Purification and characterization of two novel peptidyl toxins directed against K+ channels from the venom of new world scorpions. Biophys J. 1991;59:78. Abstract.
21.
Toshe N. Calcium sensitive delayed rectifier
potassium current in guinea pig ventricular cells.
Am J Physiol. 1990;258:H1200-H1207.
22. Kokubun S, Irisawa H. Effects of various intracellular Ca ion concentrations on the calcium current of guinea pig ventricular cells. Jpn J Physiol. 1984;34:599-611. [Medline] [Order article via Infotrieve]
23.
Decoursey TE, Chandy KG, Gupta S, Cahalan MD.
Mitogen induction of ion channels in murine lymphocytes.
J Gen Physiol. 1987;89:405-420.
24.
Leonard RJ, Garcia ML, Slaughter RS, Reuben JP.
Selective blockers of voltage-gated K+ channels
depolarize human T lymphocytes: mechanism of antiproliferative effect
of charybdotoxin. Proc Natl Acad Sci U S A. 1992;89:10094-10098.
25.
Lin CS, Boltz RC, Blake JT, Nguyen M, Talento A,
Fischer PA, Springer MS, Sigal NH, Slaughter RS, Garcia ML, Kaczorowski
J, Koo GC. Voltage-gated potassium channels regulate
calcium-dependent pathways involved in human T lymphocyte
activation. J Exp Med. 1993;177:637-645.
26. Bednarek MA, Bugianesi RM, Leonard RJ, Felix JP. Chemical synthesis and structure function studies of margatoxin, a potent inhibitor of voltage-dependent potassium channel in human T lymphocytes. Biochem Biophys Res Commun. 1994;198:619-625. [Medline] [Order article via Infotrieve]
27.
Garcia-Calvo M, Leonard RJ, Novick J, Stevens SP,
Schmalhofer W, Kaczorowski GJ, Garcia ML. Purification,
characterization, and biosynthesis of margatoxin, a component
of Centruroides margaritatus venom that selectively
inhibits voltage-dependent potassium channels. J
Biol Chem. 1993;268:18866-18874.
28. Russel JH, Dubos CB. Accelerated 86Rb (K+) release from cytotoxic T lymphocytes is a physiologic event associated with delivery of the lethal hit. J Immunol. 1983;131:1138-1141. [Abstract]
29. Schlichter LN, Sidell N, Hagiwara S. Potassium channels mediate killing in human natural killer cells. Proc Natl Acad Sci U S A. 1986;137:1650-1658.
30. Lefever A, Liepins A, Truit R. Role of K+ ion channels in lymphokine-activated killer (LAK) cell lytic function. Immunopharmacol Immunotoxicol. 1989;11:571-582. [Medline] [Order article via Infotrieve]
31. Schlichter LC, MacCourbey IC. Interactive effects of Na and K in killing by human natural killer cells. Exp Cell Res. 1989;184:99-108. [Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  G. Panyi, G. Vamosi, Z. Bacso, M. Bagdany, A. Bodnar, Z. Varga, R. Gaspar, L. Matyus, and S. Damjanovich Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells PNAS, February 3, 2004; 101(5): 1285 - 1290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sasayama, A. Matsumori, and Y. Kihara New insights into the pathophysiological role for cytokines in heart failure Cardiovasc Res, June 1, 1999; 42(3): 557 - 564. [Full Text] [PDF]
|
|
|
|
 |
|
 B. Felzen, M. Shilkrut, H. Less, I. Sarapov, G. Maor, R. Coleman, R. B. Robinson, G. Berke, and O. Binah Fas (CD95/Apo-1)–Mediated Damage to Ventricular Myocytes Induced by Cytotoxic T Lymphocytes From Perforin-Deficient Mice : A Major Role for Inositol 1,4,5-Trisphosphate Circ. Res., March 9, 1998; 82(4): 438 - 450. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||