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A potassium lonophore inhibits lymphocyte capping: The role of cations in the modulation of membrane receptors
CELLULAR
IMMUNOLOGY
(1976)
26, 21-28
A Potassium lonophore Inhibits Lymphocyte Capping: The Role of Cations in the Modulation of Membrane Receptors RONALD
Cardiovascular-Pulmonary Pathology, University
P.
DANIELE
Division,
1 AND
SANDRA
K.
HOLIAN
Department of Medicine, and the Department of School of Medicine, Philadelphia, Pa. 19104
of Pennsylvania
Received April 19,1976 Valinomycin inhibited cap formation induced by antibodies to Ig-molecules in human lymphocytes. At the concentration of valinomycin used in this study the inhibitory effects : (1) could not be explained by impaired cell viability, (2) were reversed by washing the drug from the medium and (3) were prevented by increasing the concentration of K’ in the external medium. These results suggest that valinomycin may act, in part, by altering the electrical properties of the cell membrane and suggest a role for membrane potential and cation permeability and flux in the modulation of membrane receptors.
Bridging of membrane immunoglobulin (Ig) 2 by anti-Ig molecules results in passive aggregation of surface Ig into microscopically visible patches; this is followed by active movement of the Ig aggregates to one pole of the lymphocyte (capping) ; finally, pinocytosis of the aggregate occurs with temporary disappearance of surface immunoglobulin (l-4). A similar phenomenon occurs when bivalent antigen binds surface immunoglobulin of antigen reactive cells (Z), and it has been suggested that the phenomena of patching and capping are initial steps that lead to the proliferation of B lymphocytes and the ultimate production of antibodyproducing cells. Capping can be inhibited by low temperature (4”(Z), by metabolic inhibitors (e.g., sodium azide) and by agents such as cytochalasin B which disrupt the submembranous microfilament apparatus (5). We now report that capping of the anti Ig-Ig complexes on human lymphocytes can be inhibited by valinomycin, an ionophore with high specificity for potassium. Furthermore, evidence of partial reversibility of the effect of valinomycin by increasing potassium concentration of the external medium suggests that membrane potential and/or cation fluxes may play a role in the capping phenomenon. MATERIALS
AND
METHODS
Lymphocyte Preparation Peripheral venous blood was collected in heparin from healthy volunteers. Lymphocytes (PBL) were purified from venous blood using a modification of the 1Dr. Daniele is the recipient of a Young Investigator Award from the National Heart and Lung Institute, HL-17221. This work was also supported by a USPHS .grant, CA-15822. antiserum to a Abbreviations used : Ig, Immunoglobulin ; FL-ab, Fluorescein-conjugated human Ig; HBSS, Hank’s Balanced Salt Solution. 21 1976 by Academic Press Inc. %i?$&%t:‘, * P reproduction in any form’ reserved.
22
DANIELE
AND
HOLIAN
Ficoll-Hypaque technique which included preincubation with carbonyl iron to remove monocytes (6). Lymphocytes ( > 97%) were suspended at a concentration of 5 X lo6 cells/ml in Hank’s balanced salt solution (HBSS) containing lo/O fetal calf serum. Incubation with Valinomycin Samples (1.0 ml) of this cell suspension were incubated for 30 min at 22°C with a range of valinomycin concentration ( 1O-5-1O-g 1M). Valinomycin was dissolved in 100% ethanol; the concentration of ethanol to which cells were exposed did not exceed 0.1%. Viability after exposure to all concentrations of valinomycin was tested by the trypan blue exclusion test and by cell counting (Coulter) using a modification of the cetrimide technique (7). Viability was always greater than 95%. In additional experiments, lymphocytes were cultured for 72 hr in the presence of lo-’ M valinomycin. There was no significant decrease in viability compared to similarly cultured, untreated lymphocytes. Lymphocyte
Capping
After treatment with valinomycin, aliquots (0.3 ml) of treated and untreated lymphocytes were incubated at 4°C for 30 min with 120 pg/ml of fluoresceinconjugated antiserum to human immunoglobulins (FL-ab) (Meloy) as previously described (6). After an additional 15, 30 or 60 min incubation at 22”C, the cells were fixed by adding an equal volume of 2.5% paraformaldehyde. The cells were then washed twice in phosphate-buffered saline (PBS), resuspended in glycerolPBS (1: 1) and several drops of the cell suspension mounted on slides with paraffin-sealed cover slips. The cells were then examined with a Leitz Orthoplan microscope equipped with Ploem incident illumination. Cap formation was defined by the accumulation of FL-ab to one hemisphere of the cell. Cation Chunges in the Medium In certain experiments to test the effects of monovalent cations, lymphocytes were suspended in HBSS supplemented with KC1 or choline chloride; in other experiments, equimolar concentrations of KC1 replaced NaCl in the external medium. In all experiments described below, the external medium contained 1.3 mM Ca2+ and 1.0 mM Mg 2+,0.3 mM HP02-4, 1.3 mM HsPO-4, 14.3 mM HCOs-, and 5.6 mM glucose ; the pH was between 7.2-7.4. Lymphocytes were equilibrated for 60 min at 22°C in these various solutions prior to capping experiments with or without 10-r M valinomycin being present in the external medium throughout the procedure. Potassium concentration of the final solutions was determined by flame photometry. Osmolality of the solutions was determined by melting point depression in a millisomometer. RESULTS Effect of Va&zomycin on Capping Figure 1 illustrates the inhibitory effect of various concentrations of valinomycin on cap formation of human blood lymphocytes in a representative experiment. Seven to 21% of PBL lymphocytes were positive for surface Ig and in seven
A K
IONOPHORE
INHIBITS
CAPPING
23
FIG. 1. Inhibitory effect of valinomycin on cap formation of human lymphocytes. Lymphocytes were incubated with fluorescein-conjugated antisera to human immunoglobulins (FL-ah) for 60 min at 22T. Cap formation was defined by the accumulation of FL-ab to one hemisphere of the cell. Seven to twenty-one percent of lymphocytes were positive for surface immunoglobulins. Shaded area represents range for seven experiments.
experiments, 78%-98% of the positive staining (FL-ab) cells formed caps after incubation with antiserum for 60 min. There was a marked reduction in cap formation 40 the level of 4%40% for lymphocytes treated with valinomycin at 10T5 M to lO+ M. Valinomycin at lo-’ M always resulted in a 50% or greater reduction of cap formation. Inhibition of capping was not observed at a valinomycin concentration of lo-@ M. In contrast to the typical polar “cap” in control cells (Fig. 2b), lymphocytes inhibited by the ionophore failed to cap and instead had a fluorescent pattern of multiple small patches evenly distributed over the cell surface (Fig. 2~). Valinomycin
Compared to 0 ther Inhibitors
of Capping
In order to compare further the effect of valinomycin with that of other known inhibitors of cap formation, lymphocytes were exposed to cytochalasin B (20 pg/ml), sodium azide (0.01 M) or valinomycin (lo+ M) for 30 min at 22°C. Untreated cells were incubated at 22°C as a control, and also at 4°C to demonstrate the effect of low temperature. The results are summarized in Fig. 3. Valinomycin inhibited cap formation to a greater degree than did cytochalasin B or sodium azide at the concentrations used. Complete inhibition of both patching and capping occurred at 4°C (Fig. 2a). Uncapped cells incubated in the presence of valinomycin gave a similar patching pattern as was observed in experiments involving cells treated with cytochalasim B or a metabolic inhibitor, sodium azide (Fig. 2~). Reversibility
of the Inhibitory
Eflects of Valinonzycin
To determine whether the inhibitory effect of valinomycin was reversible, cells were incubated (30 min, 22°C) with 10mT M ionophore, washed twice in large volumes of HBSS and then incubated in HBSS for 60 min at 22°C. After this recovery period, the cells were exposed to fluorescein-conjugated antisera as described above. As shown in Table 1 and Fig. 2d restoration of capping was observed for valinomycin-treated cells which had been washed and allowed to
24
DANIELE
AND
HOLIAN
FIG. 2. Cap formation of human lymphocytes. The distribution of FL-ab is shown for : (a) Lymphocytes incubated at 4°C for 60 min. A homogeneous pattern of fluorescent staining is present over most of the cell surface. (b) Cap formation for cells incubated at 22°C for 60 min with FL-ab. (c) Discrete patches of FL-ab are distributed evenly over the lymphocyte surface for cells incubated in the presence of valinomycin (lo-’ M) , sodium azide (0.01 M) or cytochalasin B (20 pg/ml). (d) Restoration of cap formation for lymphocytes treated with 10-’ M valinomycin, washed twice in HBSS, allowed to recover for 60 min at 22°C in HBSS. Cells were then treated with FL-ab as described. (e) Cap formation for cells incubated in presence of lo-’ M vahnomycin with 50 mM KC1 added to HBSS.
FIG. 3. Lymphocytes were incubated for 15, 30 and 60 min at 22°C (or 4°C) with FL-ab O--control ; OO-20 fig/ml cytoin the presence or absence of various drugs. l chalasin B ; n ---A-O.01 M sodium azide; A -A-10’ M valinomycin; O----O -cells incubated at 4°C in the absence of drugs.
A
K
IONOPHORE
INHIBITS
TABLE Reversibility
of Inhibition
2.5
CAPPING
1
of Lymphocyte
Capping by Valinomycin Caps % Exp. # 1
Exp. #2
86 40
90 17
68
86
Control Valinomycin treated cells0 Cells treated with valinomycin washed in HBSS*
a Cells incubated with 10v7 M valinomycin (22”C, 30 min), prior to capping procedure. * After incubation with 10-T M valinomycin (22”C, 30 min), cells were washed X2 HBSS and allowed to recover (22”C, 60 min). Cells were then tested for capping as described.
recover ,for 60 min at 22°C. At lO+‘M valinomycin be reversed.
the inhibitory
effects could not
Effect of Adding KC1 on Capping The inhibitory effects of valinomycin on capping were partly abolished by adding KC1 to HBSS so as to increase the K+ concentration of the suspending medium to the levels indicated in Fig, 4. The maximum effect occurred at 50 mM K+ restoring the proportion of capped cells to 80% of controls (Fig. 2e). Increasing the external K+ concentration beyond this level did not increase the proportion of capped cells in the presence of valinomycin. In separate experiments in which the NaCl of the suspending medium was replaced by equimolar concentrations of KC1 over a broad range (25 mM to 142 m&l), the inhibitory effects of valinomycin were not reversed. A representative experiment in which 50 mM of KC1 replaced NaCl is shown in Table 2. Efect of Osnzolality on Capp&g To determine whether the restoration of capping in the presence of valinomycin was related to the increase in osmolality caused by the added KCl, additional experiments were performed in which an impermeable cation, choline chloride, was added to the external medium, As indicated in Table 2, the addition of 50 m&f of choline chloride to the external medium did not override the inhibitory effects of
70 Z 60 g so s 40 30 20 IO 0 cmrRoL
+Ymy
WI.+ VA!.+ SmU K 26mM K 6%i+K
FIG. 4. Effect of increasing external K+ on the inhibition indicate mean of three experiments. Shaded bars indicate treated with 10-’ M valinomycin.
lo”,‘K
of capping by valinomycin. Bars experiments in which cells were
26
DANIELE
AND
TABLE
HOLIAN
2
Effect of Cations on Lymphocyte
Control (HBSS - 5.3 m&f K+) HBSS + 50 mM KCP HBSS + 50 mM choline Clb Valinomycin (lo-’ M) Valinomycin + 50 mM KC1 (equimolar) Valinomycin (HBSS + 50 mM KCl) Valinomycin (HBSS + 50 m&f choline Cl) a Osmolarity b Osmolarity
Capping Caps %
Rings %
84 90 89 22 21 72 14
16 10 11 78 79 28 86
equalled 380 milliosmoles. equalled 368 milliosmoles ; similar results obtained for 70 mM choline chloride.
valinomycin, nor did it alter the proportion of capped cells compared to controls when added to a cell suspension lacking valinomycin. Similar results were obtained when choline chloride was added in concentrations of 25 mM and 70 mM to treated and untreated cells. The osmolality of HBSS supplemented with 50 mM KC1 was 380 milliosmoles and did not impair cell viability (970/o viable) nor did it decrease the proportion of capped cells compared to controls (Fig. 4, Table 2). Eflect of Other Uncouplers on Capping In additional experiments, the effect on capping of 2,4-dinitrophenol (DNP), an uncoupler of oxidative metabolism, was also compared to valinomycin. Capping was inhibited to a lesser degree (” 30%) compared to valinomycin and only at a concentration (1 X 1O-3 M) which resulted in a significant impairment of cell viability (2 707 o viable). Similar observations have been reported for the inhibition of capping by DNP for mouse lymphocytes (8). DISCUSSION These findings indicate that valinomycin can reversibly inhibit the capping and pinocytosis of membrane Ig of human lymphocytes. The results cannot be explained by changes in viability, as cells which were cultured in the presence of valinomycin (10-r M) for up to 72 hr showed no significant decrease in viability over similarly cultured, untreated cells. Recently, Rutishauser et al. (9) reported that valinomycin at 1O-6 A4 inhibited morphologic changes and capping of mouse lymphocytes bound to nylon fibers. They, however, did not observe reversibility and ascribed the effect to an impairment of cellular metabolism. At the concentration of valinomycin used in this study, the inhibitory effects on capping might be explained by changes in the electrical properties of the cell membrane, ( 10, 11) or by its potential effects as an uncoupler of mitochondrial oxidative phosphorylation (12-14), or both. The possibility that the inhibitory actions of valinomycin on lymphocyte capping may be due in part to its effects on the cell membrane is supported by ( 1) the relatively poor inhibition of capping by DNP, a known uncoupler of oxidative phosphorylation ; (2) the lack of toxicity of valinomycin in short-term culture ; and, more significantly by the blocking of the effects of valinomycin by increasing potassium concentration of the external medium.
A
K
IONOPHORE
INHIBITS
CAPPING
27
Valinomycin is a cationic ionophore which selectively translocates potassium across natural and artificial membranes (15, 16). In excitable tissues, such as nerve and muscle, where the effects of this drug have been more extensively studied, valinomycin increases K+ permeability and ,may result in hyperpolarization and alteration of the electrical properties of the cell membrane (17-19). Application of the Goldman constant field equation (20, 21) predicts that these effects of valinomycin on the resting membrane potential could be reversed by increasing the potassium concentration of the external medium. This has been demonstrated experimentally in both excitable (22) and non-excitable cells (23). Recent studies have shown that local anesthetic agents, such as lidocaine, which also alter the electrical behavior of the cell membrane, inhibit cap formation of anti-Ig complexes on lymphocytes (24). Both valinomycin and lidocaine, in certain cells, can reduce membrane cation conductance (10, 25, 26). Taken together, these various observations and the present findings raise the possibility that movement of Ig-ligands within the lymphocyte membrane may be linked to membrane potential and the permeability and movement of ions across the membrane. From these considerations, our working hypothesis is that activation of receptorligand movement in the lymphocyte membrane is triggered by an alteration in membrane potential and perhaps ion permeability which may involve both potassium movement out of ‘the cell and the movement of calcium ions from the environment or membrane sites into the cytoplasm. The movement or redistribution of Ca2+ from membrane sites or bound pools (i.e. mitochondria) rather than Ca2+ influx from the external environment, is favored by the studies of Schreiner and Unanue (27). They demonstrated a Ca2+ sensitive contractile system involved in cap formation which did not require Ca2+ in the external medium. By analogy, the initiating mechanism for lymphocyte capping and perhaps subsequent B-cell proliferation, might resemble the membrane depolarization and associated ion fluxes observed in excitable tissues. The perturbing signal in the case of lymphocytes, initiating the ion flux, would involve sufficient cross-linking of membrane receptors. This pattern of ion movement (calcium in, potassium out) has been observed in several systems, including the exocytosis of granular proteins from leukocytes stimulated from leukocidin (28) and in studies where the calcium ionophore (A23187) has simulated the action of epinephrine on alpha adrenergic receptors of parotid cells (29). It is possible that part of these ion currents may also involve sodium, and thus might explain why a reduction (equimolar for K+) in the external sodium concentration in the present experiments was not associated with a reversal of the inhibition of capping by valinomycin. Thus, providing the lymphocyte membrane is relatively impermeable to sodium, ,decreases in the concentration of sodium in the external medium would not be expected to influence the resting potential, but could make a significant contribution to depolarization currents. Indeed, recent evidence indicates that the release of Ca2+ into the cytosol from bound cellular pools, a critical event in the exocytic release of neurotransmitters and hormones from excitable an’d nonexcitable cells (30, 31), may be triggered by an increase in intracellular Na+ (32). Our results suggest that valinomycin, as well as other ionophores, might be used as experimental probes in examining the effect of ion distribution and ion fluxes on receptor ligand movement within the membrane. These observations with valinomycin may also permit some general speculations about the ability of naturally
28
DANIELE
AND
HOLIAN
occurring substances ,to influence membrane potential, ion fluxes and the movement of macromolecules in the cell membrane. Ionophores have now been isolated from other cell systems (33, 34) and naturally occurring molecules with properties similar to valinomycin could play a regulatory function in various physiological processes involving modulation of membrane receptors on lymphocytes and other cells. ACKNOWLEDGMENTS We wish to thank Peter C. Nowell and David T. Rowlands, Jr., for many valuable discussions and Alfred P. Fishman for review of this manuscript. Supported in part by a Young Investigator Award from the National Heart and Lung Institute (HL-17221) and USPHS grant 15822.
REFERENCES 1. Taylor, R. B., Duffus, W. P. H., Raff, M. C., and de Petris, S., Nature New Biol. 233, 225, 1971. 2. Loor, F., Fomi, L., and Pernis, B., Eur. J. Immunol. 2, 203, 1972. 3. Unanue, E. R., Perkins, W. D., and Karnovsky, M. J., J. Exp. Med. 136, 885, 1972. 4. de Pet&, S., and Raff, M. C., Eur. J. Zmmunol. 2, 523, 1972. 5. Wessels, N. K., Spooner, B. S., Ash, J. F., Bradley, M. O., Luduena, M. A., Taylor, E. L., Wrenn, J. T., and Yamada, K. M., Science (Washington, D.C.) ,171, 135, 1971. 6. Daniele, R. P., Altose, M. D., and Rowlands, D. T., Jr., J. Cl&. Invest. 56, 986, 1975. 7. Stewart, C. C., and Ingram, M., Blood 29,628, 1967. 8. Unanue, E. R., Karnovsky, M. J., and Engers, H. D., J. Exp. Med. 137, 675, 1973. 9. Rutishauser, U., Yahara, I., and Edelman, G. M., Proc. Nat. Acad. Sci. U.S.A. 71, 1149, 1974. 10. Spector, I., Palfrey, C., and Littauer, U. Z., Nature 254, 121, 1975. 11. Romeo, D., Zabucchi, G., Miani, N., and Rossi, F., Nature 253, 542, 1975. 12. Mueller, P., and Rudin, D. O., Biochem. Biophys. Res. Commun. 26, 398, 1967. 13. Moore, C., and Pressman, B. C., Biochem Biaphys. Res. Commun. 15, 562, 1964. 14. Green, D. E., and Reible, S., Proc. Natl. Acad. Sci. U.S.A. 72, 253, 1975. 15. Pressman, B. C., Fed. Proc. 27, 1283, 1968; Fed. Proc. 32, 1698, 1973. 16. Eisenman, G., Fed. Proc. 27, 1249, 1968. 17. Podleski, T., and Changeux, J. P., Nature 221, 541, 1969. 18. Silman, I., J. Gen. Physio?. 54, 2658, 1969. 19. Hinkle, M., and Van Der Kloot, W., Camp. Biochem. Physn’ol. MA, 269, 1973. 20. Goldman, D. E., J. Gen. Physiol. 27, 37, 1943. 21. Hodgkin, A. L., and Katz, B., J. Physiol. (London) 108, 37, 1949. 22. Conway, E. J., Physiol. Rev. 37, 84, 1957. 23. Hoffman, J. F., and Laris, P. C., 1. Physiol. (London) 239, 519, 1974. 24. Ryan, G. B., Unanue, E. R., and Karnovsky, M. J., Nature 250, 56, 1974. 25. Blaustein, M. P., and Goldman, D. E., Science 153, 429, 1966. 26. Covino, B. G., New Engl. J. Med. 286, 975, 1972. 27. Schreiner, G. F., and Unanue, E. R., J. Exp. Med. 143, 15, 1976. 28. Woodin, A. M., Is “Lysozymes in Biology and Pathology” (J. T. Dingle, Ed.), Vol. 3, pp. 395-422. North Holland, Amsterdam, 1973. 29. Selinger, Z., Eimerl, S., and Schramm, M., Proc. Natl. Acad. Sci. U.S.A. 71, 128, 1974. 30. Katz, B., “The Release of Neural Transmitter Substances.” Thomas, Springfield, Illinois, 1969. 31. Rasmussen, H., Science 170, 404, 1970. 32. Lowe, D. A., Richardson, B. P., Taylor, P., and Donatsch, P., Nature 260, 337, 1976. 33. Shamoo, A. E., and Albers, R. W., Proc. Natl. Acad. Sci. U.S.A. 70, 1191, 1973. 34. Blondin, G. A., Biochem. Biophys. Res. Commun. !j6, 97, 1974.
IMMUNOLOGY
(1976)
26, 21-28
A Potassium lonophore Inhibits Lymphocyte Capping: The Role of Cations in the Modulation of Membrane Receptors RONALD
Cardiovascular-Pulmonary Pathology, University
P.
DANIELE
Division,
1 AND
SANDRA
K.
HOLIAN
Department of Medicine, and the Department of School of Medicine, Philadelphia, Pa. 19104
of Pennsylvania
Received April 19,1976 Valinomycin inhibited cap formation induced by antibodies to Ig-molecules in human lymphocytes. At the concentration of valinomycin used in this study the inhibitory effects : (1) could not be explained by impaired cell viability, (2) were reversed by washing the drug from the medium and (3) were prevented by increasing the concentration of K’ in the external medium. These results suggest that valinomycin may act, in part, by altering the electrical properties of the cell membrane and suggest a role for membrane potential and cation permeability and flux in the modulation of membrane receptors.
Bridging of membrane immunoglobulin (Ig) 2 by anti-Ig molecules results in passive aggregation of surface Ig into microscopically visible patches; this is followed by active movement of the Ig aggregates to one pole of the lymphocyte (capping) ; finally, pinocytosis of the aggregate occurs with temporary disappearance of surface immunoglobulin (l-4). A similar phenomenon occurs when bivalent antigen binds surface immunoglobulin of antigen reactive cells (Z), and it has been suggested that the phenomena of patching and capping are initial steps that lead to the proliferation of B lymphocytes and the ultimate production of antibodyproducing cells. Capping can be inhibited by low temperature (4”(Z), by metabolic inhibitors (e.g., sodium azide) and by agents such as cytochalasin B which disrupt the submembranous microfilament apparatus (5). We now report that capping of the anti Ig-Ig complexes on human lymphocytes can be inhibited by valinomycin, an ionophore with high specificity for potassium. Furthermore, evidence of partial reversibility of the effect of valinomycin by increasing potassium concentration of the external medium suggests that membrane potential and/or cation fluxes may play a role in the capping phenomenon. MATERIALS
AND
METHODS
Lymphocyte Preparation Peripheral venous blood was collected in heparin from healthy volunteers. Lymphocytes (PBL) were purified from venous blood using a modification of the 1Dr. Daniele is the recipient of a Young Investigator Award from the National Heart and Lung Institute, HL-17221. This work was also supported by a USPHS .grant, CA-15822. antiserum to a Abbreviations used : Ig, Immunoglobulin ; FL-ab, Fluorescein-conjugated human Ig; HBSS, Hank’s Balanced Salt Solution. 21 1976 by Academic Press Inc. %i?$&%t:‘, * P reproduction in any form’ reserved.
22
DANIELE
AND
HOLIAN
Ficoll-Hypaque technique which included preincubation with carbonyl iron to remove monocytes (6). Lymphocytes ( > 97%) were suspended at a concentration of 5 X lo6 cells/ml in Hank’s balanced salt solution (HBSS) containing lo/O fetal calf serum. Incubation with Valinomycin Samples (1.0 ml) of this cell suspension were incubated for 30 min at 22°C with a range of valinomycin concentration ( 1O-5-1O-g 1M). Valinomycin was dissolved in 100% ethanol; the concentration of ethanol to which cells were exposed did not exceed 0.1%. Viability after exposure to all concentrations of valinomycin was tested by the trypan blue exclusion test and by cell counting (Coulter) using a modification of the cetrimide technique (7). Viability was always greater than 95%. In additional experiments, lymphocytes were cultured for 72 hr in the presence of lo-’ M valinomycin. There was no significant decrease in viability compared to similarly cultured, untreated lymphocytes. Lymphocyte
Capping
After treatment with valinomycin, aliquots (0.3 ml) of treated and untreated lymphocytes were incubated at 4°C for 30 min with 120 pg/ml of fluoresceinconjugated antiserum to human immunoglobulins (FL-ab) (Meloy) as previously described (6). After an additional 15, 30 or 60 min incubation at 22”C, the cells were fixed by adding an equal volume of 2.5% paraformaldehyde. The cells were then washed twice in phosphate-buffered saline (PBS), resuspended in glycerolPBS (1: 1) and several drops of the cell suspension mounted on slides with paraffin-sealed cover slips. The cells were then examined with a Leitz Orthoplan microscope equipped with Ploem incident illumination. Cap formation was defined by the accumulation of FL-ab to one hemisphere of the cell. Cation Chunges in the Medium In certain experiments to test the effects of monovalent cations, lymphocytes were suspended in HBSS supplemented with KC1 or choline chloride; in other experiments, equimolar concentrations of KC1 replaced NaCl in the external medium. In all experiments described below, the external medium contained 1.3 mM Ca2+ and 1.0 mM Mg 2+,0.3 mM HP02-4, 1.3 mM HsPO-4, 14.3 mM HCOs-, and 5.6 mM glucose ; the pH was between 7.2-7.4. Lymphocytes were equilibrated for 60 min at 22°C in these various solutions prior to capping experiments with or without 10-r M valinomycin being present in the external medium throughout the procedure. Potassium concentration of the final solutions was determined by flame photometry. Osmolality of the solutions was determined by melting point depression in a millisomometer. RESULTS Effect of Va&zomycin on Capping Figure 1 illustrates the inhibitory effect of various concentrations of valinomycin on cap formation of human blood lymphocytes in a representative experiment. Seven to 21% of PBL lymphocytes were positive for surface Ig and in seven
A K
IONOPHORE
INHIBITS
CAPPING
23
FIG. 1. Inhibitory effect of valinomycin on cap formation of human lymphocytes. Lymphocytes were incubated with fluorescein-conjugated antisera to human immunoglobulins (FL-ah) for 60 min at 22T. Cap formation was defined by the accumulation of FL-ab to one hemisphere of the cell. Seven to twenty-one percent of lymphocytes were positive for surface immunoglobulins. Shaded area represents range for seven experiments.
experiments, 78%-98% of the positive staining (FL-ab) cells formed caps after incubation with antiserum for 60 min. There was a marked reduction in cap formation 40 the level of 4%40% for lymphocytes treated with valinomycin at 10T5 M to lO+ M. Valinomycin at lo-’ M always resulted in a 50% or greater reduction of cap formation. Inhibition of capping was not observed at a valinomycin concentration of lo-@ M. In contrast to the typical polar “cap” in control cells (Fig. 2b), lymphocytes inhibited by the ionophore failed to cap and instead had a fluorescent pattern of multiple small patches evenly distributed over the cell surface (Fig. 2~). Valinomycin
Compared to 0 ther Inhibitors
of Capping
In order to compare further the effect of valinomycin with that of other known inhibitors of cap formation, lymphocytes were exposed to cytochalasin B (20 pg/ml), sodium azide (0.01 M) or valinomycin (lo+ M) for 30 min at 22°C. Untreated cells were incubated at 22°C as a control, and also at 4°C to demonstrate the effect of low temperature. The results are summarized in Fig. 3. Valinomycin inhibited cap formation to a greater degree than did cytochalasin B or sodium azide at the concentrations used. Complete inhibition of both patching and capping occurred at 4°C (Fig. 2a). Uncapped cells incubated in the presence of valinomycin gave a similar patching pattern as was observed in experiments involving cells treated with cytochalasim B or a metabolic inhibitor, sodium azide (Fig. 2~). Reversibility
of the Inhibitory
Eflects of Valinonzycin
To determine whether the inhibitory effect of valinomycin was reversible, cells were incubated (30 min, 22°C) with 10mT M ionophore, washed twice in large volumes of HBSS and then incubated in HBSS for 60 min at 22°C. After this recovery period, the cells were exposed to fluorescein-conjugated antisera as described above. As shown in Table 1 and Fig. 2d restoration of capping was observed for valinomycin-treated cells which had been washed and allowed to
24
DANIELE
AND
HOLIAN
FIG. 2. Cap formation of human lymphocytes. The distribution of FL-ab is shown for : (a) Lymphocytes incubated at 4°C for 60 min. A homogeneous pattern of fluorescent staining is present over most of the cell surface. (b) Cap formation for cells incubated at 22°C for 60 min with FL-ab. (c) Discrete patches of FL-ab are distributed evenly over the lymphocyte surface for cells incubated in the presence of valinomycin (lo-’ M) , sodium azide (0.01 M) or cytochalasin B (20 pg/ml). (d) Restoration of cap formation for lymphocytes treated with 10-’ M valinomycin, washed twice in HBSS, allowed to recover for 60 min at 22°C in HBSS. Cells were then treated with FL-ab as described. (e) Cap formation for cells incubated in presence of lo-’ M vahnomycin with 50 mM KC1 added to HBSS.
FIG. 3. Lymphocytes were incubated for 15, 30 and 60 min at 22°C (or 4°C) with FL-ab O--control ; OO-20 fig/ml cytoin the presence or absence of various drugs. l chalasin B ; n ---A-O.01 M sodium azide; A -A-10’ M valinomycin; O----O -cells incubated at 4°C in the absence of drugs.
A
K
IONOPHORE
INHIBITS
TABLE Reversibility
of Inhibition
2.5
CAPPING
1
of Lymphocyte
Capping by Valinomycin Caps % Exp. # 1
Exp. #2
86 40
90 17
68
86
Control Valinomycin treated cells0 Cells treated with valinomycin washed in HBSS*
a Cells incubated with 10v7 M valinomycin (22”C, 30 min), prior to capping procedure. * After incubation with 10-T M valinomycin (22”C, 30 min), cells were washed X2 HBSS and allowed to recover (22”C, 60 min). Cells were then tested for capping as described.
recover ,for 60 min at 22°C. At lO+‘M valinomycin be reversed.
the inhibitory
effects could not
Effect of Adding KC1 on Capping The inhibitory effects of valinomycin on capping were partly abolished by adding KC1 to HBSS so as to increase the K+ concentration of the suspending medium to the levels indicated in Fig, 4. The maximum effect occurred at 50 mM K+ restoring the proportion of capped cells to 80% of controls (Fig. 2e). Increasing the external K+ concentration beyond this level did not increase the proportion of capped cells in the presence of valinomycin. In separate experiments in which the NaCl of the suspending medium was replaced by equimolar concentrations of KC1 over a broad range (25 mM to 142 m&l), the inhibitory effects of valinomycin were not reversed. A representative experiment in which 50 mM of KC1 replaced NaCl is shown in Table 2. Efect of Osnzolality on Capp&g To determine whether the restoration of capping in the presence of valinomycin was related to the increase in osmolality caused by the added KCl, additional experiments were performed in which an impermeable cation, choline chloride, was added to the external medium, As indicated in Table 2, the addition of 50 m&f of choline chloride to the external medium did not override the inhibitory effects of
70 Z 60 g so s 40 30 20 IO 0 cmrRoL
+Ymy
WI.+ VA!.+ SmU K 26mM K 6%i+K
FIG. 4. Effect of increasing external K+ on the inhibition indicate mean of three experiments. Shaded bars indicate treated with 10-’ M valinomycin.
lo”,‘K
of capping by valinomycin. Bars experiments in which cells were
26
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2
Effect of Cations on Lymphocyte
Control (HBSS - 5.3 m&f K+) HBSS + 50 mM KCP HBSS + 50 mM choline Clb Valinomycin (lo-’ M) Valinomycin + 50 mM KC1 (equimolar) Valinomycin (HBSS + 50 mM KCl) Valinomycin (HBSS + 50 m&f choline Cl) a Osmolarity b Osmolarity
Capping Caps %
Rings %
84 90 89 22 21 72 14
16 10 11 78 79 28 86
equalled 380 milliosmoles. equalled 368 milliosmoles ; similar results obtained for 70 mM choline chloride.
valinomycin, nor did it alter the proportion of capped cells compared to controls when added to a cell suspension lacking valinomycin. Similar results were obtained when choline chloride was added in concentrations of 25 mM and 70 mM to treated and untreated cells. The osmolality of HBSS supplemented with 50 mM KC1 was 380 milliosmoles and did not impair cell viability (970/o viable) nor did it decrease the proportion of capped cells compared to controls (Fig. 4, Table 2). Eflect of Other Uncouplers on Capping In additional experiments, the effect on capping of 2,4-dinitrophenol (DNP), an uncoupler of oxidative metabolism, was also compared to valinomycin. Capping was inhibited to a lesser degree (” 30%) compared to valinomycin and only at a concentration (1 X 1O-3 M) which resulted in a significant impairment of cell viability (2 707 o viable). Similar observations have been reported for the inhibition of capping by DNP for mouse lymphocytes (8). DISCUSSION These findings indicate that valinomycin can reversibly inhibit the capping and pinocytosis of membrane Ig of human lymphocytes. The results cannot be explained by changes in viability, as cells which were cultured in the presence of valinomycin (10-r M) for up to 72 hr showed no significant decrease in viability over similarly cultured, untreated cells. Recently, Rutishauser et al. (9) reported that valinomycin at 1O-6 A4 inhibited morphologic changes and capping of mouse lymphocytes bound to nylon fibers. They, however, did not observe reversibility and ascribed the effect to an impairment of cellular metabolism. At the concentration of valinomycin used in this study, the inhibitory effects on capping might be explained by changes in the electrical properties of the cell membrane, ( 10, 11) or by its potential effects as an uncoupler of mitochondrial oxidative phosphorylation (12-14), or both. The possibility that the inhibitory actions of valinomycin on lymphocyte capping may be due in part to its effects on the cell membrane is supported by ( 1) the relatively poor inhibition of capping by DNP, a known uncoupler of oxidative phosphorylation ; (2) the lack of toxicity of valinomycin in short-term culture ; and, more significantly by the blocking of the effects of valinomycin by increasing potassium concentration of the external medium.
A
K
IONOPHORE
INHIBITS
CAPPING
27
Valinomycin is a cationic ionophore which selectively translocates potassium across natural and artificial membranes (15, 16). In excitable tissues, such as nerve and muscle, where the effects of this drug have been more extensively studied, valinomycin increases K+ permeability and ,may result in hyperpolarization and alteration of the electrical properties of the cell membrane (17-19). Application of the Goldman constant field equation (20, 21) predicts that these effects of valinomycin on the resting membrane potential could be reversed by increasing the potassium concentration of the external medium. This has been demonstrated experimentally in both excitable (22) and non-excitable cells (23). Recent studies have shown that local anesthetic agents, such as lidocaine, which also alter the electrical behavior of the cell membrane, inhibit cap formation of anti-Ig complexes on lymphocytes (24). Both valinomycin and lidocaine, in certain cells, can reduce membrane cation conductance (10, 25, 26). Taken together, these various observations and the present findings raise the possibility that movement of Ig-ligands within the lymphocyte membrane may be linked to membrane potential and the permeability and movement of ions across the membrane. From these considerations, our working hypothesis is that activation of receptorligand movement in the lymphocyte membrane is triggered by an alteration in membrane potential and perhaps ion permeability which may involve both potassium movement out of ‘the cell and the movement of calcium ions from the environment or membrane sites into the cytoplasm. The movement or redistribution of Ca2+ from membrane sites or bound pools (i.e. mitochondria) rather than Ca2+ influx from the external environment, is favored by the studies of Schreiner and Unanue (27). They demonstrated a Ca2+ sensitive contractile system involved in cap formation which did not require Ca2+ in the external medium. By analogy, the initiating mechanism for lymphocyte capping and perhaps subsequent B-cell proliferation, might resemble the membrane depolarization and associated ion fluxes observed in excitable tissues. The perturbing signal in the case of lymphocytes, initiating the ion flux, would involve sufficient cross-linking of membrane receptors. This pattern of ion movement (calcium in, potassium out) has been observed in several systems, including the exocytosis of granular proteins from leukocytes stimulated from leukocidin (28) and in studies where the calcium ionophore (A23187) has simulated the action of epinephrine on alpha adrenergic receptors of parotid cells (29). It is possible that part of these ion currents may also involve sodium, and thus might explain why a reduction (equimolar for K+) in the external sodium concentration in the present experiments was not associated with a reversal of the inhibition of capping by valinomycin. Thus, providing the lymphocyte membrane is relatively impermeable to sodium, ,decreases in the concentration of sodium in the external medium would not be expected to influence the resting potential, but could make a significant contribution to depolarization currents. Indeed, recent evidence indicates that the release of Ca2+ into the cytosol from bound cellular pools, a critical event in the exocytic release of neurotransmitters and hormones from excitable an’d nonexcitable cells (30, 31), may be triggered by an increase in intracellular Na+ (32). Our results suggest that valinomycin, as well as other ionophores, might be used as experimental probes in examining the effect of ion distribution and ion fluxes on receptor ligand movement within the membrane. These observations with valinomycin may also permit some general speculations about the ability of naturally
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occurring substances ,to influence membrane potential, ion fluxes and the movement of macromolecules in the cell membrane. Ionophores have now been isolated from other cell systems (33, 34) and naturally occurring molecules with properties similar to valinomycin could play a regulatory function in various physiological processes involving modulation of membrane receptors on lymphocytes and other cells. ACKNOWLEDGMENTS We wish to thank Peter C. Nowell and David T. Rowlands, Jr., for many valuable discussions and Alfred P. Fishman for review of this manuscript. Supported in part by a Young Investigator Award from the National Heart and Lung Institute (HL-17221) and USPHS grant 15822.
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