Mar 31, 1986 - ABSTRACT. Mature human T lymphocytes proliferate in response to the mitogen phytohemagglutinin (PHA), but im- mature thymocytes lacking ...
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 5625-5629, August 1986 Immunology
K channels are expressed early in human T-cell development (patch-clamp recording/human thymocytes/T lymphocytes/mitogenic responses/immune competence)
LYANNE SCHLICHTER*t, NEIL SIDELLt, AND SUSUMU HAGIWARA* *Department of Physiology and Jerry Lewis Neuromuscular Research Center, and tDivision of Surgical Oncology, University of California, Los Angeles, CA 90024
Contributed by Susumu Hagiwara, March 31, 1986
Mature human T lymphocytes proliferate in ABSTRACT response to the mitogen phytohemagglutinin (PHA), but immature thymocytes lacking the T3 receptor (T3- thymocytes) do not. Because functioning K channels are required for proliferation of mature T cells, we asked whether immunoincompetent T3- thymocytes lack normal K channels. We report that T3- thymocytes have a K+ current similar to that of mature peripheral T cells-that is, similar voltage dependence, activation and inactivation kinetics, and pharmacology. Moreover, the maximal specific K+ conductance is the same for both cell types, implying a similar density of activable channels in each cell. In assessing the functional responses of the channels to PHA, we found that the K+ current ofimmature and mature cells responds similarly to the mitogen. Responses near the threshold voltage for activating the K+ current were variable; the K+ conductance and rate of activation were increased, decreased, or unchanged after PHA treatment. For several cells, the voltage dependence of the conductance and activation kinetics was shifted in opposite directions. At more positive voltages, PHA consistently caused a 10-20% suppression of conductance that was not due to the addition of an inward current, to changes in the time course of activation or inactivation, or to changes in the steady-state level of inactivation. The effects of PHA on the K+ current cannot be explained by a simple shift in surface potential, as has been hypothesized to be involved in its triggering of T-cell proliferation. Taken together, our findings show (i) K channels are expressed very early in T-cell differentiation, possibly before thymic processing, (i) differential responses of the K+ current to PHA do not account for the failure of T3- thymocytes to proliferate, and (iii) changes in surface potential are probably not a necessary early event in activation of T cells by PHA.
ture thymocytes is either a lack of K channels or failure of the channels to respond appropriately to mitogenic stimuli.
METHODS Cell Preparation. Human thymocytes were obtained from children undergoing cardiac surgery who required partial thymus resection to facilitate surgical exposure. Thymocyte suspensions were enriched for immature cells by exploiting their lack of reactivity to OKT3 (Ortho Diagnostics) monoclonal antibody [T3- cells (thymocytes lacking the T3 receptor)], using an indirect "panning" technique as described by Reinherz et al. (10). Based on indirect immunofluorescence, this procedure yielded a T3- thymocyte population of >95% purity compared with 80-90% T3- cells in the unenriched population. Mature T lymphocytes were isolated from peripheral blood mononuclear cells obtained by standard density-gradient centrifugation on Ficoll-Isopaque, followed by depletion of plastic- and nylon-wool-adherent B cells and monocytes. The purity of T3+ cells (mature T cells) was >95%, as determined by indirect immunofluorescence using the OKT3 antibody. Solutions. Phytohemagglutinin (PHA) was freshly mixed from stock solutions to a final concentration of 10 ,ug/ml in standard recording solution (see below). The mitogenic activity of the PHA was confirmed by its ability to induce blastogenesis of peripheral blood lymphocytes in a standard 3-day assay as described (11). The standard bathing solution contained (in mM) 150 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 D-glucose, 10 Hepes, and 4 NaOH (pH 7.4). The pipette solution contained (in mM) 100 KF, 20 NaCl, 1 MgCl2, 15 D-glucose, 10 KHepes, and S K2EGTA (pH 7.4). All electrical recordings were made at 20-22°C. Electrophysiology. After a high-resistance (>20 GQl) seal was formed between a patch pipette of 3-8 MQl resistance, the patch was ruptured by light suction to establish a whole-cell recording. Cell input resistances were >10 Gfl. The membrane potential was held at -95 mV in a chamber continuously perfused with standard bathing solution (details in ref. 7). Junction potentials were measured and corrections were made as in previous work (12).
With the recent development of the Gil-seal patch-clamp technique (1) the small cells of the immune system have become accessible to detailed electrophysiological study. It has been found that K channels are the predominant ion channels in human and mouse T cells and T-cell lines (2-5), in cultured macrophages (6), and in human natural killer cells (7, 8). The K channels appear to be necessary for immune function in these cells, whether it is proliferation of T cells (2, 9) or lysis of target cells by natural killer cells (7, 8). A central role for the K channels in the activation of T lymphocytes was recently proposed because drugs that block the K channels block early events in mitogenesis, such as interleukin 2 synthesis/secretion, and because mitogenic stimulation caused an immediate change in the channel gating characteristics (2, 9). The importance of these K channels for proliferation of mature T lymphocytes and the reported effects of mitogens on the K+ current in these cells (2, 5) led us to ask whether one defect in immunoincompetent, imma-
RESULTS Currents in Immature Human Thymocytes. Highly enriched preparations of immature human thymocytes (>95% T3-) were obtained as described in Methods. Currents were first recorded with standard solution in a continuously perfused bath. Fig. 1 shows membrane currents recorded from an immature (T3-) human thymocyte, using the wholecell configuration of the patch-clamp technique. The membrane was held at -95 mV and stepped in 10-mV increments Abbreviations: PHA, phytohemagglutinin; T3- thymocytes, thymocytes lacking the T3 receptor. tPresent address: Department of Physiology, University of Toronto, ON, Canada M5S 1A8.
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for 60 ms to test potentias ltwen -65 and +55 mV. For clarity, only traces at -55, -4!, -35, -15, and +5 mV are shown. At -45 mV an inward current appeared, which increased as the voltage increased to +5 mV and then decreased at more positive membrane potentials. This inward current showed the fast activation and inactivation characteristic of Na+ currents of excitable cells. Evidence that this is a Na+ current is that it was completely blocked by 0.6 ,uM tetrodotoxin and that the direction of the current reversed from inward to outward at +45 mV, which is very close to the calculated Na equilibrium potential of +52 mV. Na+ currents were seen in a minority of thymocytes (5 of 28) and in none of the peripheral T lymphocytes. At -45 mV a time-dependent outward current appeared and increased in amplitude as the voltage was made more positive. Evidence that the outward current is carried by K+ is that the direction of the tail current reversed very close to the calculated equilibrium potentil of -81 mV in all eight cells examined, and with eqil K; cocentrations inside and outside the cell the reversal pential was about 0 mV. When the K+ current was blocked by 4u4miopyridine (10 mM), (0.1 M) there was little or quinidine (0.1 mM), or verap no remaining current. These reis uest that voltage-sensitive Ca2+ currents were absopt or were below the detection limits of 1-2 pA in our whole-cell recordings. However, this observation does not rule out the possibility of non-voltagegated Ca channels. Such currents would have been subtracted as part of the time-independent leakage current. The K+ currents in all 28 thymocytes examined were qualitatively similar to those in Fig. 1 (see also Fig. 2A). Activation was seen at about -50 mV and showed a sigmoid time course. Inactivation was seen at about -15 mV and above as a decrease in current with time after the peak. Inactivation was much slower than activation at all voltages and was relatively insensitive to voltage above -15 mV; therefore, the peak current during a pulse was very close to the maximal activated value. Cumulative inactivation was seen between pulses that were separated by 100 ms) compared with activation (ti12 < 6 ms) that the peak current was reduced by -35 mV) the conductance was suppressed 10-15% by PHA (Fig. 3B) and activation was consistently slower (Fig. 3D). The suppression of K+ conductance by PHA was not reversible during up to 30 min of washing in standard solution. During each treatment and wash period the maximal K+ conductance remained fairly constant; hence, there was no significant "run-down" of current during the experiment.
DISCUSSION It was recently proposed that K channels in mature T lymphocytes play a necessary role in mitogenesis and that an early activation event in these cells is an increase in the K+ current (2, 9). Therefore, it seemed that a contributory factor to lack of immune competence of immature thymocytes might be a lack of K channels or a defect in the response of these channels during immune stimulation. We have now ruled out these possibilities as a principal cause of immunoincompetence. Our results show that immature, immunoincompetent (T3-) thymocytes have a K+ current similar in all respects to that in mature peripheral T cells. Immature and mature cells have the same maximal specific K+ conductance, implying a similar channel density. Thus, the development of the lymphocyte K channel does not coincide with acquisition of T-cell immunocompetence. Furthermore, we have shown that PHA, which is mitogenic for mature T cells but not for T3- thymocytes, affects the K+ current similarly in both cell types. Near the threshold voltage the response was highly variable; the K+ current was increased, decreased, or unchanged. A more consistent result was a significant reduction (10-20%) in K+ current at more positive voltages in immature and mature cells. The only difference we observed between the response to PHA of mature T cells and immature thymocytes was that the
activation time (ti,2) was slowed after PHA treatment in mature T cells at voltages at which the K+ current was suppressed. Overall, our results indicate that differential responses of the current do not account for the failure of immature thymocytes to proliferate after PHA treatment. It has been suggested that PHA changes the surface potential of T cells such that more K channels open and that this effect might be a necessary early step in activation (2, 13). Our results do not support this contention nor are the effects of PHA on the currents consistent with a simple shift in surface potential since (i) little or no shift in the g-V relation was seen near threshold, (ii) for several cells the voltage dependence of the conductance and of the activation kinetics shifted in opposite directions, and (iii) the g-V relations
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before and after PHA treatment did not converge on a common maximal conductance-instead, gm,, was suppressed 10-20% by PHA. A possible explanation for this suppression comes from the recent finding that intracellular Ca2' reduces the K+ conductance of human T cells (16). Since PHA causes intracellular Ca2+ to rise in T cells (17) and in thymocytes (18), the decrease in K+ conductance we observe after PHA treatment might reflect increased intracellular Ca2+ levels despite the presence of KF in the pipette solution. It has also been suggested that the T3 receptor of mature T cells may be physically or functionally related to a Ca channel (19). A similar relationship between the K channel and the T3 receptor can be ruled out since T3- thymocytes and human natural killer cells, which also lack the T3 receptor, have the same type of K channel (7, 8). Finally, our finding that all of the T3- thymocytes examined possess similar K channels indicates that the development of these channels occurs either before or very early during thymic processing. Prethymic development is further suggested by the presence of this channel in natural killer cells, lymphocytes that share some T-cell surface markers but whose development usually does not involve a thymic residency (20). Thus, this type of K channel, displaying kinetics and voltage sensitivity similar to delayed rectifier K channels but an unusual sensitivity to Ca-channel blockers, probably develops before the divergence of discrete T-cell and natural killer cell lineages. Further studies on ion channels in other types of immune cells, such as normal myeloid cells and B lymphocytes, will help determine the tissue and lineage specificity of this type of K channel. Such information will be useful for helping to elucidate the roles that these ion channels play in immune activation and effector-phase events. We thank Gloria Sze for excellent technical assistance. This work supported by National Institutes of Health Grants CA30515 and NS09012, a Muscular Dystrophy Association grant, and National Institutes of Health Training Grant NS07101.
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