Immunopharmacology, 22 (1991) 85-92 © 1991 Elsevier Science Publishers B.V. All rights reserved 0162-3109/91/$03.50
85
IMPHAR 00556
Does veraparnil act as an immunomodulatory drug in vivo ? Alan S. Maisel 1,2, Dave Murray 1 Susan Polizzi 1,2, Harvey J. Motulsky 1, Otto-Erich B r o d d e 3, Lambertus J.H. van Tits 3 and Martin C. Michel 1,3 ~Departments of Medicine and Pharmacology, University of California, San Diego, La Jolla, CA, 2Veterans Administration Hospital La Jolla, CA, U.S.A., and 3Department of Medicine, University of Essen, Essen, F.R.G. (Received 20 June 1991; accepted 27 June 1991)
Abstract: Based on in vitro data, previous investigators have hypothesized that Ca2 + entry blockers might affect lymphocyte activation, proliferation and effector function. We have tested this hypothesis by comparing the in vitro and in vivo effects ot the Ca 2+ entry blocker verapamil on human lymphocytes. In vitro high concentrations of verapamil (100/~M) inhibited mitogen-stimulated Ca2 ÷ influx, inositol phosphate generation, and proliferation; similar effects were observed with diltiazem and nifedipine. In vivo treatment of healthy volunteers with verapamil (2-4 times 240 mg per day for 7 days) did not affect the number of circulating lymphocytes or their subset distribution. Moreover, we did not observe any effect of in vivo treatment with verapamil on mitogen-stimulated lymphocyte proliferation or expression of interleukin-2 receptors in vitro. We conclude that the inhibitory effects of verapamil on lymphocyte activation in vitro are unlikely to be of therapeutic relevance and may not be related to the Ca2 ÷ entry blocking effects of this drug.
Key words:
Verapamil; Interleukin-2; Nifedipine; Diltiazem; Lymphocyte proliferation; Calcium
Introduction
The development of an immune response to a given antigen requires the clonal proliferation of resting lymphocytes responsive to that particular antigen. The first step of this clonal proliferation is the transition of responsive lymphocytes from the Go state into the cell cycle (Gardner, 1989; Miyajima et al., 1988; Hadden, 1988; Cooper, 1987; Steel and Hutchins, 1989; Cambier et al. 1987). In in vitro studies, this process is frequently mimicked by the use of polyclonal activators such as the plant lectin concanavalin A (Con A). Various studies have demonstrated that
Correspondence: M.C. Michel, Nephrol. Lab. IG 1, Klinikum, Hufelandstr. 55, D-4300 Essen 1, F.R.G.
increases in lymphocyte intracellular C a 2 + and activation of a phospholipase C are important biochemical events which can be observed in the very early phase of the activation of resting lymphocytes (Hadden, 1988; Gelfand etal., 1987; Linch etal., 1987; DeFranco etal., 1987; Cambier et al., 1987). The idea that such increases in intracellular Ca 2 + and activation of a phospholipase C (with subsequent stimulation of a protein kinase C) are important for the activation of resting lymphocytes is supported by the finding that a combination of Ca 2 + ionophore and phorbol ester (directly activating a protein kinase C) can stimulate resting lymphocytes from the Go phase into the cell cycle (Nobrega et al., 1986; Procopio et al., 1988). Moreover, it has recently been demonstrated that the dihydropyridine Bay K 8644 (which activates certain Ca 2 + channels)
86 can also activate resting lymphocytes (Young et al., 1988). Therefore, it is not surprising that various investigators have reported inhibitory effects of organic Ca 2 + entry blockers on the activation of resting lymphocytes in vitro (Kimball et al., 1988; Gelfand et al., 1986; Chandy et al., 1984; Nobrega et al., 1986; Dos Reis etal., 1988). Based on these findings it has been speculated that C a 2 + entry blockers may also have immunomodulatory effects in vivo (Young et al., 1988). Since the C a 2 + entry blocker concentrations used in the in vitro studies were quite high we have tested this hypothesis by comparing the in vitro effects of verapamil and other Ca 2+ entry blockers with their in vivo effects. Specifically we determined the concentration-dependency of the in vitro inhibition of mitogen-stimulated Ca 2 + influx, inositol phosphate generation and proliferation for verapamil, diltiazem and nifedipine. These data were contrasted with those obtained after treating healthy human volunteers with the Ca 2 + entry blocker verapamil for one week and assessing various parameters of immunity. Materials and Methods Proliferation studies Sterilely prepared peripheral blood mononuclear leukocytes (mostly lymphocytes) were resuspended in RPMI medium supplemented with 25 #g/ml gentamycin, 2 mM glutamine, and 20~o fetal calf serum at a final density of 106 cells/ml. 200/~1 of cell suspension were transferred to each well of 96-well flat bottom plates. In some wells Con A (4.5, 9, 18, or 36/~g/ml final) was present. Cells were incubated for 4 days in a humidified incubator with 95 ~o air/5 ~o CO2. Additionally, verapamil was present in some experiments in the indicated concentrations. During the last 16 h of a 96-h incubation, 2/~Ci of [3H]thymidine (dissolved in 20 #1 of RPMI) were added to each well. At the end of the incubation the cells were harvested onto G F / C filters. The filters were washed with 10 ml of water, placed into vials, 4.5 ml of scintillator were added, and the incorporated
radioactivity was quantified in a liquid scintillation counter at 42 ~o efficiency. Each data point was assessed in quadruplicate in each experiment. For the initial in vitro studies lymphocytes were obtained from drug-free young healthy volunteers. Measurements of intracellular Ca 2 + We have previously described our methods for determination of the free intracellular Ca 2 + concentration in great detail (Motulsky and Michel, 1988). Briefly, cells were loaded with the fluorescent indicator dye Fura-2, washed twice, and resuspended in buffer. Subsequently fluorescence was determined at room temperature with excitation at 340nm and emission at 510 nm. Our calculation for the conversion of raw fluorescence data into intracellular Ca 2 + concentrations included correction factors for the amount of extracellular dye and for dilution by the added drugs. In experiments involving nifedipine we also corrected for the quench caused by this yellow compound. Measurements of inositol phosphate generation Mitogen-stimulated accumulation of inositol phosphates was determined as previously described (van Tits et al., 1991). Briefly, cells were loaded with [3H]myoinositol, washed, and thereafter incubated with mitogen in the presence or absence of the Ca 2 ÷ entry blockers for 30 min. The incubation was terminated by addition of chloroform/methanol, and the formed inositol phosphates were isolated from the aqueous phase by elution from Dowex anion exchange columns. In vivo studies Twelve healthy volunteers (mean age: 27.6 (22-42) years, 8 males/4 females) participated in the in vivo part of our study after having given informed written consent. The study protocol had been approved by the University of California San Diego committee for Studies Involving Human Subjects. The volunteers were given 240 mg slow release capsules of verapamil (Knoll Pharmaceuticals, Whippany, N J) for 7 days; 5 subjects received 240 mg twice daily and 7 sub-
87 jects received 240 mg four times daily. Systolic blood pressure did not drop more than 10 mmHg during treatment in any subject. Before treatment and 12 h after the last drug intake venous blood was withdrawn for the preparation of mononuclear leukocytes according to published procedures (DeBlasi et al., 1986). All subjects rested for 20 min in the sitting position before venipuncture. Our initial analysis did not reveal any significant differences between values observed in the two treatment groups for any parameters measured (data not shown). Therefore, we have pooled the data from both treatment groups for the sake of brevity in presentation.
Determination of subsets of circulating lymphocytes We assessed the number of cells in each subset by flow-cytometric analysis as recently described (Maisel et al., 1990). Briefly, monoclonal antibodies were directly conjugated with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) and we used a combination of antiLeu 3a FITC and anti-Leu 2a PE or anti-Leu 7 FITC and anti-Leu 2a PE with two-color immunofluorescence, or anti Leu 4 FITC or antiLeu 11 FITC with single-color immunofluorescence. A negative control consisting of IgG~ FITC and IgG2 PE was included for each set of cells stained. Five thousand cells were analyzed per sample. Electronic 'gating' of the lymphocyte population was performed based on forward and side-scatter parameters. This method yielded the relative proportion of each subset as a percentage of the total lymphocytes counted. The absolute number of cells in each subset was calculated by multiplying the fraction of cells in each subset times the absolute lymphocyte count derived from the white blood cell and differential count (S plus IV Coulter, Hialeah, FL). IL-2 receptor expression Sterilely prepared MNL were suspended in supplemented RPMI medium (see above) in the absence and presence of Con A (18 #g/ml). After 24, 48 or 72 h the cells were centrifuged. The expression of IL-2 receptors was determined
fluorimetrically using anti-CD25 antibodies as described above (Maisel et al., 1991).
Chemicals [3H]myo-inositol (spec. activity 15-20 Ci/mmol) was purchased from New England Nuclear (Dreieich, F.R.G.), Fura-2 from Molecular Probes (Eugene, OR), Con A, verapamil, diltiazem, nifedipine and all other chemicals were from Sigma (St, Louis, MO). For the in vitro studies, stock solutions of the Ca 2 + entry blockers were prepared in ethanol; in these experiments it was ascertained that the maximal final ethanol concentration in the assay did not affect the results by itself (data not shown). Data analysis Differences between groups were tested for statistical significance by paired two-tailed t-tests; a Pvalue of less than 0.05 was considered to be significant. Differences found in the in vivo studies were not statistically significant for any parameter studied whether or not corrections for multiple comparisons were made. All data presented are mean + SEM of n determinations.
Results
In vitro studies Verapamil (100#M) partially inhibited the increased in intracellular Ca 2 + and the generation of inositol phosphates elicited by Con A or PHA (Fig. 1). Similarly, the same concentration of diltiazem and nifedipine also inhibited the Con Astimulated Ca 2 + increases and inositol phosphate generation (Table I). Lower concentrations of all three drugs had only minor inhibitory effects which could not reliably be quantified (data not shown). Verapamil also inhibited the Con Astimulated proliferation of human lymphocytes as assessed by [3H]thymidine uptake (Fig. 2). This effect was observed only at a concentration of 100 # M whereas lower concentrations had no significant effects. Nevertheless, this does not appear to be a non-specific toxic effect since
88
IO0/~M verapamil did not affect the basal [3H]thymidine uptake. x
c
n=6
rl = 4
75
75 °
~g
x:~
2_
+
50 °
50
u
In vivo studies K g
25 D
o 251
PHA
Con A
PHA
Con
A
Fig. 1. Effects ofverapamil on Con A- and PHA-stimulated Ca 2 + increases and inositol phosphate generation in vitro. Ca z+ influx was stimulated by 100 #g/ml of Con A or PHA, and inositol phosphate generation was stimulated by 18 #g/ml of Con A or 1.8 #g/ml of PHA. The inhibitory effect of verapamil (100 #M) was quantified in each experiment and expressed as % inhibition of the response to the mitogens in the absence of verapamil. Data are mean + SEM of the indicated number of experiments.
Treatment of healthy subjects with verapamil did not affect the total white blood cell count, the relative or absolute number of circulating lymphocytes (Table II) or the subset distribution of circulating lymphocytes (Fig. 3). Additional parameters were assessed to determine whether the functional responsiveness of circulating lymphocytes might be altered by verapamil treatment.
TABLE I Effects of Ca 2 + entry blockers on concanavalin A-stimulated Ca 2 + increases and inositol phosphate generation
c EO[1[FOi × + verapamil 4~
~-
300000
~
1 ~M
o + verapama] lO ,~M [] + vePapam]] 100 ,~M rq = 4
200000
Verapamil Diltiazem Nifedipine
~ooooo
1o 20 [concanava]in A],
30 ~g/m]
40
Fig. 2. Effects of verapamil on Con A-stimulated proliferation of human lymphocytes in vitro.
% Inhibition of Con A-stimulated inositol phosphate generation
% Inhibition of Con A-stimulated Ca 2 + influx
50 + 6 25 + 4 78 + 2
34 +_ 4 24 + 3 14 + 4
Cells were stimulated with 100 #g/ml (Ca 2+ experiments) or 18 #g/ml (inositol phosphate experiments) of Con A in the presence and absence of the Ca 2 + entry blockers, which were all tested at a concentration of 100/~M. Data are mean + SEM of 3-6 experiments.
TABLE II Effects of verapamil treatment on circulating white blood cells
WBC (cells//21) Lymphocytes (cells//~l) Lymphocytes (% of WBC) The,per (cells//~l) Tsupp . . . . . . /cytotoxi~(cells//~1) Thelper/Ysupp . . . . . . /cytotoxic ratio
Before treatment
After treatment
6750 2412 38.3 1088 587 1.96
7050 2128 31.5 959 491 2.08
+ 688 _+ 150 _+ 3.4 + 89 + 62 + 2.08
+ 569 +_ 111 _+ 2.3 + 86 _+ 56 _+ 0.17
Delta - 300 + 284 + 6.8 + 129 + 96+ 0.13 +
395 156 3.6 138 55 0.24
All parameters were quantified prior to and after 7 days of treatment with 2-4 times 240 mg of verapamil daily. Values are mean + SEM of 10 subjects.
89
~ l b e f o r e treatment
75
J~after
x:
400,000
treatment 300,000
#
50
:
200,000
co
,g
o before treatment * after treatment n = 10
4~
%
100,000
25
a~ 0
o
5
10 15 20 ~5 30 [eencanavalin A], ~g/m]
35
Fig. 4. Effects of a 7-day treatment with verapamil on the Con A-stimulated proliferation of human lymphocytes. Fig. 3. Effects of a 7-day treatment with verapamil on the subset distribution of circulating mononuclear leukocytes in healthy volunteers.
The Con A-stimulated lymphocyte proliferation in vitro was not significantly different in cells obtained prior to or after verapamil treatment (Fig. 4). Moreover, neither the basal nor the Con A-stimulated expression of interleukin-2 receptors was altered by verapamil treatment (Table III).
TABLE III Effects of verapamil treatment on interleukin-2 receptor expression Before treatment
After treatment
Delta
After 24 h basal Con A-treated
3_+0 41 + 6
6+ 1 48 + 1
-2+ 8 +
A f t e r 48 h basal Con A-treated
4+ 1 56 + 8
5+0 64 + 8
-1 + 1 8 + 11
A f t e r 72 h basal Con A-treated
4_+ 1 79 + 4
6_+ 1 79 + 3
-2_+ 0 +
1 6
2 7
Cells were obtained prior to treatment and after 7 days of treatment with 2 - 4 times 240 mg of verapamil daily, incubated in medium and treated with either 18 #g/ml Con A or vehicle (basal). At the indicated times, cells were harvested and the percentage of cells expressing interleukin-2 receptors was determined using appropriate fluorescent antibodies. Data are mean + SEM of 3 - 5 subjects, since data before and after treatment could not be obtained from each subject for technical reasons.
Discussion
The role of increases in intracellular free Ca 2+ and activation of a protein kinase C in the activation of resting lymphocytes is well established (Hadden, 1988; Gelfand et al., 1987; Linch et al., 1987; DeFranco etal., 1987; Cambier etal., 1987). Since the increase in intracellular C a 2+ comes predominantly if not exclusively from influx of extracellular Ca 2 + rather than mobilization from intracellular stores (Chen et al., 1986; Gelfand et al., 1987; Linch et al., 1987; DeFranco et al., 1987; Cambier et al., 1987; van Tits et al., 1991), it has been speculated whether organic C a 2 + entry blockers might exert immunomodulatory effects in vivo (Young et al., 1988). This hypothesis has been tested in the present study. Our data confirm previous observations (Kimball etal., 1988; Gelfand etal., 1986; Chandy et al., 1984; Nobrega et al., 1986; Dos Reis et al., 1988) that Ca 2+ entry blockers can inhibit lymphocyte activation in vitro as assessed by biochemical markers such a s C a 2+ influx, inositol phosphate generation and [ 3H ]thymidine uptake. Additionally it has been shown that verapamil can inhibit the mitogen-stimulated accumulation of IL-6 m R N A (Walz et al., 1990). However, the required concentrations (100 gM) are considerably higher than those required for the inhibition of L-type Ca 2 + channels and human lymphocytes lack L-type Ca 2+ channels (Kuno et al., 1986). Thus it is questionable whether the inhibitory effects of the C a 2 + entry blockers are
90 related to Ca 2 ÷ channel blocking activity or to some other effect. It has been proposed therefore, that the inhibitory in vitro effects of C a 2 + entry blockers on lymphocytes may be related to their effects on K + rather than Ca 2+ channels (Chandy et al., 1984; Nobrega et al., 1986; Dos Reis et al., 1988). The high concentrations of Ca 2 + entry blockers required for in vitro inhibitory effects obviously make an in vivo role in the regulation of lymphocyte function unlikely for this group of drugs. Moreover, it could be argued that any major immunomodulatory effect of C a 2 + entry blockers should have been detected previously considering the long and widespread use of these drugs. On the other hand, it is always hard to extrapolate in vitro concentration-response relationships to in vivo settings. For example, only micromolar concentrations of fi-adrenergic agonists can inhibit lymphocyte activation in vitro (Feldman et al., 1987; van Tits et al., 1991) but therapeutic doses of the fi-adrenergic agonist as well as physiological plasma levels of endogenous catecholamines can induce lymphopenia in vivo (Maisel et al., 1990). Such regulation may affect subsets of lymphocytes in a distinct manner because they may differ in their sensitivity to the respective drug (Maisel et al., 1989). Thus, the lymphopenia induced by fi-adrenergic agonists selectively affects T-lymphocytes (Maisel et al., 1990); similarly, antagonism of the endogenous catecholamines by the fl-adrenergic antagonist propranolol selectively increases the number of circulating T-lymphocytes without affecting those of other lymphocyte subsets (Maisel et al., 1991). These considerations stimulated us to determine the in vivo effects of verapamil on the number of circulating lymphocytes and their subsets. Our study did not detect any alterations of the number of circulating lymphocytes or other white blood cells or their ex vivo proliferative responsiveness to Con A. Moreover, we excluded the possibility that verapamil might selectively affect certain lymphocyte subsets which might be exceptionally sensitive to C a 2+ entry verapamil. We also did not detect any alterations of the above
parameters in 6 healthy subjects treated with 60 mg t.i.d, of a sustained release diltiazem formulation or in 3 subjects treated with 60 mg/d nifedipine for 7 days each (data not shown). In conclusion, our data demonstrate that very high concentrations of organic Ca 2 + entry blockers such as verapamil may inhibit lymphocyte activation in vitro but do not affect the number or function of circulating lymphocytes in healthy volunteers in vivo. However, we would like to point to the possibility that some Ca 2+ entry blockers may nevertheless affect human immunity under selected circumstances. For example, it has been demonstrated that the Ca 2 + entry blocker diltiazem can reduce the allograft rejection incidence in renal transplant patients (Wagner et al., 1986). However, it is not clear whether this immuno-inhibitory effect is caused by its Ca 2+ entry blocking action or may be solely attributed to a pharmacokinetic interaction of diltiazem with the metabolism and excretion of cyclosporin A (Kunzendorf et al., 1987). Further studies on a possible immunomodulatory effect of Ca 2 + entry blockers in vivo in more complex settings, such as co-administration with other immunomodulatory drugs, appear to be necessary.
Acknowledgements We thank Dr. Ann Rearden for flow cytometric analysis and the respective pharmaceutical companies for their gifts of drugs. M.C.M. was the recipient of a fellowship of the Deutsche Forschungsgemeinschaft.
References Cambier JC, Justement LB, Newell MK, Chen ZZ, Harris LK, Sandoval VM, Klemsz MJ, Ransom JT. Transmembrane signals and intracellular 'second messengers' in the regulation of quiescent B-lymphocyte activation. Immunol Rev 1987; 95: 37-57. Chandy KG, Cahalan MD, McLaughlinC, Gupta S. Voltage-gated potassium channels are required for human T lymphocyte activation. J Exp Med 1984; 160: 369-385.
91 Chen ZZ, Coggeshall KM, Cambier JC. Translocation of protein kinaseC during membrane immunoglobulinmediated transmembrane signaling in B lymphocytes. J Immunol 1986; 136: 2300-2304. Cooper MD. B lymphocytes. Normal development and function. New Engl J Med 1987; 317: 1452-1456. DeBlasiA, Maisel AS, Feldman RD, Ziegler MG, Fratelli M, DiLallo M, Smith DA, Lai C-YC, Motulsky HJ. In vivo regulation offl-adrenergic receptors on human mononuclear leukocytes: assessment of receptor number, location, and function after posture change, exercise, and isoproterenol infusion. J Clin Endocrinol Metab 1986; 63: 847-853. DeFranco AL, Gold MR, Jakway JP. B-lymphocyte signal transduction in response to anti-immunoglobulin and bacterial lipopolysaccaride. Immunol Rev 1987; 95: 161-176. Dos Reis GA, N6brega AF, Persechini PM. Stage-specific distinctions in potassium channel blocker control of T-lymphocyte activation. Int J Immunopharmacol 1988; 10: 217-226. Feldman RD, Hunninghake GW, McArdle WL. fl-Adrenergic receptor-mediated suppression of interleukin 2 receptors in human lymphocytes. J Immunol 1987; 139: 3355-3359. Gardner P. Calcium and T lymphocyte activation. Cell 1989; 59: 15-20. Gelfand EW, Cheung RK, Grinstein S, Mills GB. Characterization of the role for calcium influx in mitogen-induced triggering of human T cells. Identification of calciumdependent and calcium-independent signals. Eur J Immunol 1986; 16: 907-912. Gelfand EW, Mills GB, Cheung RK, Lee JWW, Grinstein S. Transmembrane ion fluxes during activation of human T lymphocytes: role of Ca 2+, Na+/H + exchange and phospholipid turnover. Immunol Rev 1987; 95: 59-87. Hadden JW. Transmembrane signals in the activation of T lymphocytes by mitogenic antigens. Immunol Today 1988; 9: 235-239. Kimball PM, Gamaz N, Sell S. Two episodes of calcium uptake associated with T-lymphocyte activation. Cell Immunol 1988; 113: 107-116. Kuno M, GoronzyJ, Weyand CM, Gardner P. Singlechannel and whole-cell recordings of mitogen-regulated inward currents in human cloned T lymphocytes. Nature 1986; 323: 269-273. Kunzendorf U, Walz G, Neumayer H-H, Wagner K, Keller F, Offermann G. Einflul3 von Diltiazem auf die Ciclosporin-Blutspiegel. Klin Wochenschr 1987; 65: 1101-1103. Linch DC, Wallace DL, O'Flynn K. Signal transduction in
human T lymphocytes. Immunol Rev 1987; 95: 137-159. Maisel AS, Fowler P, Rearden A, Motulsky HJ, Michel MC. A new method for isolation of human lymphocyte subsets reveals differential regulation offl-adrenergic receptors by terbutaline treatment. Clin Pharmacol Ther 1989; 46: 429-439. Malsel AS, Knowlton KU, Fowler P, Rearden A, Ziegler MG, Motulsky H J, Insel PA, Michel MC. Adrenergic control of circulating lymphocyte subpopulations. Effects of congestive heart failure, dynamic exercise, and terbutaline treatment. J Clin Invest 1990; 85: 462-467. Maisel AS, Murray D, Lotz M, Rearden A, Irwin M, Michel MC. Propranolol treatment affects parameters of human immunity. Immunopharmacol 1991; in press: Miyajima A, Miyatake S, Schreurs J, De Vries J, Arai N, Yokota T, Arai K-I. Coordinate regulation of immune and inflammatory responses by T cell-derived lymphokines. FASEB J 1988; 2: 2462-2473. Motulsky H J, Michel MC. Neuropeptide Y mobilizes Ca 2 + and inhibits adenylate cyclase in human erythroleukemia cells. Am J Physiol 1988; 255: E880-E885. Nobrega AF, Maldonado MS, Dos Reis GA. Analysis ot isolated and combined effects of calcium ionophore and phorbol ester on T lymphocyte activation. Clin Exp Immunol 1986; 65: 559-569. Procopio A, Gismondi A, Paolini R, Morrone S, Testi R, Piccoli M, Frati L, Herberman RB, Santoni A. Proliferative effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophores on human large granular lymphocytes (LGL). Cell Immunol 1988; 113: 70-81. Steel CM, Hutchins D. Soluble factors and cell-surface molecules involved in human B lymphocyte activation, growth and differentiation. Biochem Biophys Acta 1989; 989: 133-151. van Tits LJH, Michel MC, Motulsky H J, Maisel AS, Brodde O-E. Cyclic AMP counteracts mitogen-induced inositol phosphate generation and increass in intracellular Ca 2 +-concentrations in human lymphocytes. Br J Pharmacol 1991; 103: 1288-1294. Wagner K, Albrecht S, Neumayer H-H. Prevention ot delayed graft function by a calcium antagonist - a randomized trial in renal graft recipients on cyclosporin. Transplant Proc 1986; 18: 1269-1271. Walz G, Zanker B, Barth C, Wieder KJ, Clark SC, Strom TB. Transcriptional modulation of human IL-6 gene expression by verapamil. J Immunol 1990; 144: 4242-4248. Young W, Chen J, Jung F, Gardner P. Dihydropyridine Bay K 8644 activates T lymphocyte calcium-permeable channels. Mol Pharmacol 1988; 34: 239-244.
85
IMPHAR 00556
Does veraparnil act as an immunomodulatory drug in vivo ? Alan S. Maisel 1,2, Dave Murray 1 Susan Polizzi 1,2, Harvey J. Motulsky 1, Otto-Erich B r o d d e 3, Lambertus J.H. van Tits 3 and Martin C. Michel 1,3 ~Departments of Medicine and Pharmacology, University of California, San Diego, La Jolla, CA, 2Veterans Administration Hospital La Jolla, CA, U.S.A., and 3Department of Medicine, University of Essen, Essen, F.R.G. (Received 20 June 1991; accepted 27 June 1991)
Abstract: Based on in vitro data, previous investigators have hypothesized that Ca2 + entry blockers might affect lymphocyte activation, proliferation and effector function. We have tested this hypothesis by comparing the in vitro and in vivo effects ot the Ca 2+ entry blocker verapamil on human lymphocytes. In vitro high concentrations of verapamil (100/~M) inhibited mitogen-stimulated Ca2 ÷ influx, inositol phosphate generation, and proliferation; similar effects were observed with diltiazem and nifedipine. In vivo treatment of healthy volunteers with verapamil (2-4 times 240 mg per day for 7 days) did not affect the number of circulating lymphocytes or their subset distribution. Moreover, we did not observe any effect of in vivo treatment with verapamil on mitogen-stimulated lymphocyte proliferation or expression of interleukin-2 receptors in vitro. We conclude that the inhibitory effects of verapamil on lymphocyte activation in vitro are unlikely to be of therapeutic relevance and may not be related to the Ca2 ÷ entry blocking effects of this drug.
Key words:
Verapamil; Interleukin-2; Nifedipine; Diltiazem; Lymphocyte proliferation; Calcium
Introduction
The development of an immune response to a given antigen requires the clonal proliferation of resting lymphocytes responsive to that particular antigen. The first step of this clonal proliferation is the transition of responsive lymphocytes from the Go state into the cell cycle (Gardner, 1989; Miyajima et al., 1988; Hadden, 1988; Cooper, 1987; Steel and Hutchins, 1989; Cambier et al. 1987). In in vitro studies, this process is frequently mimicked by the use of polyclonal activators such as the plant lectin concanavalin A (Con A). Various studies have demonstrated that
Correspondence: M.C. Michel, Nephrol. Lab. IG 1, Klinikum, Hufelandstr. 55, D-4300 Essen 1, F.R.G.
increases in lymphocyte intracellular C a 2 + and activation of a phospholipase C are important biochemical events which can be observed in the very early phase of the activation of resting lymphocytes (Hadden, 1988; Gelfand etal., 1987; Linch etal., 1987; DeFranco etal., 1987; Cambier et al., 1987). The idea that such increases in intracellular Ca 2 + and activation of a phospholipase C (with subsequent stimulation of a protein kinase C) are important for the activation of resting lymphocytes is supported by the finding that a combination of Ca 2 + ionophore and phorbol ester (directly activating a protein kinase C) can stimulate resting lymphocytes from the Go phase into the cell cycle (Nobrega et al., 1986; Procopio et al., 1988). Moreover, it has recently been demonstrated that the dihydropyridine Bay K 8644 (which activates certain Ca 2 + channels)
86 can also activate resting lymphocytes (Young et al., 1988). Therefore, it is not surprising that various investigators have reported inhibitory effects of organic Ca 2 + entry blockers on the activation of resting lymphocytes in vitro (Kimball et al., 1988; Gelfand et al., 1986; Chandy et al., 1984; Nobrega et al., 1986; Dos Reis etal., 1988). Based on these findings it has been speculated that C a 2 + entry blockers may also have immunomodulatory effects in vivo (Young et al., 1988). Since the C a 2 + entry blocker concentrations used in the in vitro studies were quite high we have tested this hypothesis by comparing the in vitro effects of verapamil and other Ca 2+ entry blockers with their in vivo effects. Specifically we determined the concentration-dependency of the in vitro inhibition of mitogen-stimulated Ca 2 + influx, inositol phosphate generation and proliferation for verapamil, diltiazem and nifedipine. These data were contrasted with those obtained after treating healthy human volunteers with the Ca 2 + entry blocker verapamil for one week and assessing various parameters of immunity. Materials and Methods Proliferation studies Sterilely prepared peripheral blood mononuclear leukocytes (mostly lymphocytes) were resuspended in RPMI medium supplemented with 25 #g/ml gentamycin, 2 mM glutamine, and 20~o fetal calf serum at a final density of 106 cells/ml. 200/~1 of cell suspension were transferred to each well of 96-well flat bottom plates. In some wells Con A (4.5, 9, 18, or 36/~g/ml final) was present. Cells were incubated for 4 days in a humidified incubator with 95 ~o air/5 ~o CO2. Additionally, verapamil was present in some experiments in the indicated concentrations. During the last 16 h of a 96-h incubation, 2/~Ci of [3H]thymidine (dissolved in 20 #1 of RPMI) were added to each well. At the end of the incubation the cells were harvested onto G F / C filters. The filters were washed with 10 ml of water, placed into vials, 4.5 ml of scintillator were added, and the incorporated
radioactivity was quantified in a liquid scintillation counter at 42 ~o efficiency. Each data point was assessed in quadruplicate in each experiment. For the initial in vitro studies lymphocytes were obtained from drug-free young healthy volunteers. Measurements of intracellular Ca 2 + We have previously described our methods for determination of the free intracellular Ca 2 + concentration in great detail (Motulsky and Michel, 1988). Briefly, cells were loaded with the fluorescent indicator dye Fura-2, washed twice, and resuspended in buffer. Subsequently fluorescence was determined at room temperature with excitation at 340nm and emission at 510 nm. Our calculation for the conversion of raw fluorescence data into intracellular Ca 2 + concentrations included correction factors for the amount of extracellular dye and for dilution by the added drugs. In experiments involving nifedipine we also corrected for the quench caused by this yellow compound. Measurements of inositol phosphate generation Mitogen-stimulated accumulation of inositol phosphates was determined as previously described (van Tits et al., 1991). Briefly, cells were loaded with [3H]myoinositol, washed, and thereafter incubated with mitogen in the presence or absence of the Ca 2 ÷ entry blockers for 30 min. The incubation was terminated by addition of chloroform/methanol, and the formed inositol phosphates were isolated from the aqueous phase by elution from Dowex anion exchange columns. In vivo studies Twelve healthy volunteers (mean age: 27.6 (22-42) years, 8 males/4 females) participated in the in vivo part of our study after having given informed written consent. The study protocol had been approved by the University of California San Diego committee for Studies Involving Human Subjects. The volunteers were given 240 mg slow release capsules of verapamil (Knoll Pharmaceuticals, Whippany, N J) for 7 days; 5 subjects received 240 mg twice daily and 7 sub-
87 jects received 240 mg four times daily. Systolic blood pressure did not drop more than 10 mmHg during treatment in any subject. Before treatment and 12 h after the last drug intake venous blood was withdrawn for the preparation of mononuclear leukocytes according to published procedures (DeBlasi et al., 1986). All subjects rested for 20 min in the sitting position before venipuncture. Our initial analysis did not reveal any significant differences between values observed in the two treatment groups for any parameters measured (data not shown). Therefore, we have pooled the data from both treatment groups for the sake of brevity in presentation.
Determination of subsets of circulating lymphocytes We assessed the number of cells in each subset by flow-cytometric analysis as recently described (Maisel et al., 1990). Briefly, monoclonal antibodies were directly conjugated with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE) and we used a combination of antiLeu 3a FITC and anti-Leu 2a PE or anti-Leu 7 FITC and anti-Leu 2a PE with two-color immunofluorescence, or anti Leu 4 FITC or antiLeu 11 FITC with single-color immunofluorescence. A negative control consisting of IgG~ FITC and IgG2 PE was included for each set of cells stained. Five thousand cells were analyzed per sample. Electronic 'gating' of the lymphocyte population was performed based on forward and side-scatter parameters. This method yielded the relative proportion of each subset as a percentage of the total lymphocytes counted. The absolute number of cells in each subset was calculated by multiplying the fraction of cells in each subset times the absolute lymphocyte count derived from the white blood cell and differential count (S plus IV Coulter, Hialeah, FL). IL-2 receptor expression Sterilely prepared MNL were suspended in supplemented RPMI medium (see above) in the absence and presence of Con A (18 #g/ml). After 24, 48 or 72 h the cells were centrifuged. The expression of IL-2 receptors was determined
fluorimetrically using anti-CD25 antibodies as described above (Maisel et al., 1991).
Chemicals [3H]myo-inositol (spec. activity 15-20 Ci/mmol) was purchased from New England Nuclear (Dreieich, F.R.G.), Fura-2 from Molecular Probes (Eugene, OR), Con A, verapamil, diltiazem, nifedipine and all other chemicals were from Sigma (St, Louis, MO). For the in vitro studies, stock solutions of the Ca 2 + entry blockers were prepared in ethanol; in these experiments it was ascertained that the maximal final ethanol concentration in the assay did not affect the results by itself (data not shown). Data analysis Differences between groups were tested for statistical significance by paired two-tailed t-tests; a Pvalue of less than 0.05 was considered to be significant. Differences found in the in vivo studies were not statistically significant for any parameter studied whether or not corrections for multiple comparisons were made. All data presented are mean + SEM of n determinations.
Results
In vitro studies Verapamil (100#M) partially inhibited the increased in intracellular Ca 2 + and the generation of inositol phosphates elicited by Con A or PHA (Fig. 1). Similarly, the same concentration of diltiazem and nifedipine also inhibited the Con Astimulated Ca 2 + increases and inositol phosphate generation (Table I). Lower concentrations of all three drugs had only minor inhibitory effects which could not reliably be quantified (data not shown). Verapamil also inhibited the Con Astimulated proliferation of human lymphocytes as assessed by [3H]thymidine uptake (Fig. 2). This effect was observed only at a concentration of 100 # M whereas lower concentrations had no significant effects. Nevertheless, this does not appear to be a non-specific toxic effect since
88
IO0/~M verapamil did not affect the basal [3H]thymidine uptake. x
c
n=6
rl = 4
75
75 °
~g
x:~
2_
+
50 °
50
u
In vivo studies K g
25 D
o 251
PHA
Con A
PHA
Con
A
Fig. 1. Effects ofverapamil on Con A- and PHA-stimulated Ca 2 + increases and inositol phosphate generation in vitro. Ca z+ influx was stimulated by 100 #g/ml of Con A or PHA, and inositol phosphate generation was stimulated by 18 #g/ml of Con A or 1.8 #g/ml of PHA. The inhibitory effect of verapamil (100 #M) was quantified in each experiment and expressed as % inhibition of the response to the mitogens in the absence of verapamil. Data are mean + SEM of the indicated number of experiments.
Treatment of healthy subjects with verapamil did not affect the total white blood cell count, the relative or absolute number of circulating lymphocytes (Table II) or the subset distribution of circulating lymphocytes (Fig. 3). Additional parameters were assessed to determine whether the functional responsiveness of circulating lymphocytes might be altered by verapamil treatment.
TABLE I Effects of Ca 2 + entry blockers on concanavalin A-stimulated Ca 2 + increases and inositol phosphate generation
c EO[1[FOi × + verapamil 4~
~-
300000
~
1 ~M
o + verapama] lO ,~M [] + vePapam]] 100 ,~M rq = 4
200000
Verapamil Diltiazem Nifedipine
~ooooo
1o 20 [concanava]in A],
30 ~g/m]
40
Fig. 2. Effects of verapamil on Con A-stimulated proliferation of human lymphocytes in vitro.
% Inhibition of Con A-stimulated inositol phosphate generation
% Inhibition of Con A-stimulated Ca 2 + influx
50 + 6 25 + 4 78 + 2
34 +_ 4 24 + 3 14 + 4
Cells were stimulated with 100 #g/ml (Ca 2+ experiments) or 18 #g/ml (inositol phosphate experiments) of Con A in the presence and absence of the Ca 2 + entry blockers, which were all tested at a concentration of 100/~M. Data are mean + SEM of 3-6 experiments.
TABLE II Effects of verapamil treatment on circulating white blood cells
WBC (cells//21) Lymphocytes (cells//~l) Lymphocytes (% of WBC) The,per (cells//~l) Tsupp . . . . . . /cytotoxi~(cells//~1) Thelper/Ysupp . . . . . . /cytotoxic ratio
Before treatment
After treatment
6750 2412 38.3 1088 587 1.96
7050 2128 31.5 959 491 2.08
+ 688 _+ 150 _+ 3.4 + 89 + 62 + 2.08
+ 569 +_ 111 _+ 2.3 + 86 _+ 56 _+ 0.17
Delta - 300 + 284 + 6.8 + 129 + 96+ 0.13 +
395 156 3.6 138 55 0.24
All parameters were quantified prior to and after 7 days of treatment with 2-4 times 240 mg of verapamil daily. Values are mean + SEM of 10 subjects.
89
~ l b e f o r e treatment
75
J~after
x:
400,000
treatment 300,000
#
50
:
200,000
co
,g
o before treatment * after treatment n = 10
4~
%
100,000
25
a~ 0
o
5
10 15 20 ~5 30 [eencanavalin A], ~g/m]
35
Fig. 4. Effects of a 7-day treatment with verapamil on the Con A-stimulated proliferation of human lymphocytes. Fig. 3. Effects of a 7-day treatment with verapamil on the subset distribution of circulating mononuclear leukocytes in healthy volunteers.
The Con A-stimulated lymphocyte proliferation in vitro was not significantly different in cells obtained prior to or after verapamil treatment (Fig. 4). Moreover, neither the basal nor the Con A-stimulated expression of interleukin-2 receptors was altered by verapamil treatment (Table III).
TABLE III Effects of verapamil treatment on interleukin-2 receptor expression Before treatment
After treatment
Delta
After 24 h basal Con A-treated
3_+0 41 + 6
6+ 1 48 + 1
-2+ 8 +
A f t e r 48 h basal Con A-treated
4+ 1 56 + 8
5+0 64 + 8
-1 + 1 8 + 11
A f t e r 72 h basal Con A-treated
4_+ 1 79 + 4
6_+ 1 79 + 3
-2_+ 0 +
1 6
2 7
Cells were obtained prior to treatment and after 7 days of treatment with 2 - 4 times 240 mg of verapamil daily, incubated in medium and treated with either 18 #g/ml Con A or vehicle (basal). At the indicated times, cells were harvested and the percentage of cells expressing interleukin-2 receptors was determined using appropriate fluorescent antibodies. Data are mean + SEM of 3 - 5 subjects, since data before and after treatment could not be obtained from each subject for technical reasons.
Discussion
The role of increases in intracellular free Ca 2+ and activation of a protein kinase C in the activation of resting lymphocytes is well established (Hadden, 1988; Gelfand et al., 1987; Linch et al., 1987; DeFranco etal., 1987; Cambier etal., 1987). Since the increase in intracellular C a 2+ comes predominantly if not exclusively from influx of extracellular Ca 2 + rather than mobilization from intracellular stores (Chen et al., 1986; Gelfand et al., 1987; Linch et al., 1987; DeFranco et al., 1987; Cambier et al., 1987; van Tits et al., 1991), it has been speculated whether organic C a 2 + entry blockers might exert immunomodulatory effects in vivo (Young et al., 1988). This hypothesis has been tested in the present study. Our data confirm previous observations (Kimball etal., 1988; Gelfand etal., 1986; Chandy et al., 1984; Nobrega et al., 1986; Dos Reis et al., 1988) that Ca 2+ entry blockers can inhibit lymphocyte activation in vitro as assessed by biochemical markers such a s C a 2+ influx, inositol phosphate generation and [ 3H ]thymidine uptake. Additionally it has been shown that verapamil can inhibit the mitogen-stimulated accumulation of IL-6 m R N A (Walz et al., 1990). However, the required concentrations (100 gM) are considerably higher than those required for the inhibition of L-type Ca 2 + channels and human lymphocytes lack L-type Ca 2+ channels (Kuno et al., 1986). Thus it is questionable whether the inhibitory effects of the C a 2 + entry blockers are
90 related to Ca 2 ÷ channel blocking activity or to some other effect. It has been proposed therefore, that the inhibitory in vitro effects of C a 2 + entry blockers on lymphocytes may be related to their effects on K + rather than Ca 2+ channels (Chandy et al., 1984; Nobrega et al., 1986; Dos Reis et al., 1988). The high concentrations of Ca 2 + entry blockers required for in vitro inhibitory effects obviously make an in vivo role in the regulation of lymphocyte function unlikely for this group of drugs. Moreover, it could be argued that any major immunomodulatory effect of C a 2 + entry blockers should have been detected previously considering the long and widespread use of these drugs. On the other hand, it is always hard to extrapolate in vitro concentration-response relationships to in vivo settings. For example, only micromolar concentrations of fi-adrenergic agonists can inhibit lymphocyte activation in vitro (Feldman et al., 1987; van Tits et al., 1991) but therapeutic doses of the fi-adrenergic agonist as well as physiological plasma levels of endogenous catecholamines can induce lymphopenia in vivo (Maisel et al., 1990). Such regulation may affect subsets of lymphocytes in a distinct manner because they may differ in their sensitivity to the respective drug (Maisel et al., 1989). Thus, the lymphopenia induced by fi-adrenergic agonists selectively affects T-lymphocytes (Maisel et al., 1990); similarly, antagonism of the endogenous catecholamines by the fl-adrenergic antagonist propranolol selectively increases the number of circulating T-lymphocytes without affecting those of other lymphocyte subsets (Maisel et al., 1991). These considerations stimulated us to determine the in vivo effects of verapamil on the number of circulating lymphocytes and their subsets. Our study did not detect any alterations of the number of circulating lymphocytes or other white blood cells or their ex vivo proliferative responsiveness to Con A. Moreover, we excluded the possibility that verapamil might selectively affect certain lymphocyte subsets which might be exceptionally sensitive to C a 2+ entry verapamil. We also did not detect any alterations of the above
parameters in 6 healthy subjects treated with 60 mg t.i.d, of a sustained release diltiazem formulation or in 3 subjects treated with 60 mg/d nifedipine for 7 days each (data not shown). In conclusion, our data demonstrate that very high concentrations of organic Ca 2 + entry blockers such as verapamil may inhibit lymphocyte activation in vitro but do not affect the number or function of circulating lymphocytes in healthy volunteers in vivo. However, we would like to point to the possibility that some Ca 2+ entry blockers may nevertheless affect human immunity under selected circumstances. For example, it has been demonstrated that the Ca 2 + entry blocker diltiazem can reduce the allograft rejection incidence in renal transplant patients (Wagner et al., 1986). However, it is not clear whether this immuno-inhibitory effect is caused by its Ca 2+ entry blocking action or may be solely attributed to a pharmacokinetic interaction of diltiazem with the metabolism and excretion of cyclosporin A (Kunzendorf et al., 1987). Further studies on a possible immunomodulatory effect of Ca 2 + entry blockers in vivo in more complex settings, such as co-administration with other immunomodulatory drugs, appear to be necessary.
Acknowledgements We thank Dr. Ann Rearden for flow cytometric analysis and the respective pharmaceutical companies for their gifts of drugs. M.C.M. was the recipient of a fellowship of the Deutsche Forschungsgemeinschaft.
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