Interaction of Cytotoxic T Lymphocytes and Guinea Pig Ventricular Myocytes
Pharmacological Modulation by Blocking K+ Currents in Cytotoxic T Lymphocytes
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.
- cytotoxic T lymphocytes
- peritoneal exudate cytotoxic C lymphocytes
- ventricular myocytes
- heart transplant rejection
- Ca2+ channel blockers
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
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.
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 MΩ 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.
In vivo primed PELs were obtained from BALB/c mice 4 to 5 days after the secondary intraperitoneal immunization with 25×106 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.
Results are expressed as mean±SEM. To compare means of two populations, we used Student’s t test for paired or unpaired observations.
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⇓.
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.
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
|APA||=||action potential amplitude|
|APD||=||action potential duration|
|APD50||=||APD at 50% repolarization|
|Con A||=||concanavalin A|
|CTL||=||cytotoxic (killer) T lymphocyte|
|ICa,L||=||L-type Ca2+ current|
|IK||=||delayed outward K+ current|
|NCS||=||newborn calf sera|
|PEL||=||peritoneal exudate CTL|
|Vm||=||resting membrane potential|
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.
- © 1996 American Heart Association, Inc.
Auchincloss HD, Sachs H. Transplantation and graft rejection. In: Paul WE, ed. Fundamental Immunology. New York, NY: Raven Press Publishers; 1989:889-922.
Binah O. Immune effector mechanisms in heart transplant rejection. Cardiovasc Res. 1994;28:1748-1757.
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.
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.
Huber SA, Morasaka A, Choate M. T cells expressing the γδ T-cell receptor potentiate coxsackievirus B3-induced myocarditis. J Virol. 1992;66:6541-6546.
Brener Z. Pathogenesis and immunopathology of chronic Chagas’ disease. Mem Inst Oswaldo Cruz. 1987;82:205-213.
Morris SA, Tanowitz HB, Wittner M, Bilezikian JP. Pathophysiological insights into the cardiomyopathy of Chagas’ disease. Circulation. 1990;82:1900-1909.
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.
Berke G. Interaction of cytotoxic T lymphocytes and target cells. Prog Allergy. 1980;27:68-133.
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.
Sharon N, Lis A. Lectins as cell recognition molecules. Science. 1989;246:227-233.
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.
Gallin EK. Ion channels in leukocytes. Pharmacol Rev. 1991;71:775-811.
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.
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.
Toshe N. Calcium sensitive delayed rectifier potassium current in guinea pig ventricular cells. Am J Physiol. 1990;258:H1200-H1207.
Decoursey TE, Chandy KG, Gupta S, Cahalan MD. Mitogen induction of ion channels in murine lymphocytes. J Gen Physiol. 1987;89:405-420.
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.
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.
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.
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.
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.