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The in vitro effect of phenytoin and carbamazepine on subpopulations of human blood mononuclear cells
Int. J. lmmunopharmac.. Vol, 5, No, 4, pp. 2 8 3 - 2 8 8 . 1983. Printed in Great Britain.
0192-0561/83 $3,00 + ,00 @ 1983 International Society for Immunopharmacology.
THE I N VITRO EFFECT OF PHENYTOIN AND CARBAMAZEPINE ON SUBPOPULATIONS OF H U M A N BLOOD MONONUCLEAR CELLS NILS ERIK G1LHUS§ Department of Neurology and Broegelmann Research Laboratory for Microbiology, University of Bergen, Bergen, Norway
(Received 25 May 1982)
Abstraet--Mononuclear cells from peripheral blood of 10 healthy blood donors were incubated with phenytoin and carbamazepine dissolved in 1°70propyleneglycol. Phenytoin 20 mg/l significantly reduced the percentage of active E rosette-forming T-lymphocytes; to mean 17.6070compared to mean 25.9070 in control incubations (P<0.05). Carbamazepine 10 mg/1 reduced this percentage to 21.0070 (0.05
Both phenytoin and carbamazepine can induce a moderate reduction of the concentrations of IgA and IgM in serum, whereas deficiency of IgA occurs in about 5 070 of phenytoin-treated patients (Aarli, 1976; Gilhus, Strandjord & Aarli, 1982b). The total numbers of circulating lymphocytes are reduced in patients taking phenytoin, while the distribution between different lymphocyte subpopulations is unchanged (Gilhus, Matte & Aarli, 1982a). However, phenytoin probably reduces the number of T-lymphocytes in vitro (Hornby & MuUen, 1977; Neilan, 1979). Also carbamazepine may reduce the total number of lymphocytes (Sorrell & Forbes, 1975). The effect of this drug on lymphocyte subpopulations has not been examined. The synthesizing activity of mitogen-stimulated lymphocytes is reduced by both drugs (Sorrell & Forbes, 1975). In the present study, various subpopulations of mononuclear blood cells were enumerated before and after treating the cells with phenytoin and carbamazepine in concentrations similar to the serum concentrations in epileptic patients taking these drugs. In some experiments the effect of various drug concentrations was examined.
EXPERIMENTAL PROCEDURES
Peripheral blood mononuclear cells Heparinized peripheral blood was obtained from 10 consecutive healthy blood donors. Mononuclear cells were isolated on Lymphoprep ® (Nyegaard & Co., Oslo, Norway), washed 3 times in isotonic phosphate-buffered saline, pH 7.2 (PBS) and adjusted to a concentration of 3 × 106 cells/ml (BOyum, 1968).
Reagents Pure phenytoin powder (batch 427180) was provided by Nyegaard & Co., and pure carbamazepine powder (batch 002979) by Ciba-Geigy Pharma A/S, StrOmmen, Norway. The selected amount of each drug was dissolved in 1°70 propyleneglycol in PBS (v/v). Before incubation, the concentrations of phenytoin and carbamazepine were controlled by a gas-chromatographic method measuring the underivatized drug (Bredesen & Johannessen, 1974). Sheep and ox blood were collected in an equal volume of Alsever's solution. Before use the erythrocytes were washed 3 times in 10 volumes of PBS and
§ Correspondence to: Dr. N. E. Gilhus, Department of Neurology, N-5016 Haukeland Sykehus, Bergen, Norway. 283
284
NILS ERIK GILHUS
packed at 1000g for l0 min (100o70).
Incubation with phenytoin, carbamazepine or solvent One millilitre of the suspension of peripheral blood mononuclear cells was centrifuged at 200 g for 5 min, and the supernatant pipetted off. The cells were then resuspended in 0.1 ml of the various incubation media: phenytoin 20 mg/1, carbamazepine 10 mg/1, 107o propyleneglycol, and PBS. Cells from 3 donors were incubated with phenytoin and with carbamazepine in concentrations of 5, 10, 20, 40 and 80 mg/1. After incubation for 30 min at 37°C the cells were washed once in PBS, and the concentration of cells was adjusted to 3 x 106/ml. The mononuclear cells were incubated with the drugs or solvents within 4 h after the blood was drawn from the donor. Rosette techniques Sheep erythrocytes (SE) and ox erythrocytes (OE) were treated as indicated below, and made up to a 1°7o suspension. Equal volumes of the suspensions of indicator cells and of the mononuclear cells (0.05 ml of each) were mixed and incubated at the time and temperature indicated below. The percentage of mononuclear cells rosetting with the indicator cells (rosette-forming cells--RFC) were estimated by counting at least 200 cells in a counting chamber. A rosetting cell was defined as a cell binding 3 or more erythrocytes. A c t i v e E RFC. Active T-lymphocytes were enumerated by mixing the isolated mononuclear cells with the untreated SE. The cells were immediately centrifuged for 5 min at 200 g, the pellets gently resuspended, and the number of rosettes counted (Smith, Kerman, Ezdinli & Stefani, 1975). Total E RFC. The total number of lymphocytes with receptors for SE (i.e. total T-lymphocytes) were enumerated according to Kaplan & Clark (1974). Sheep erythrocytes were treated with 2-aminoethyli s o t h i o u r o n i u m bromide hydrobromide (AET) (Sigma Chemical Co., St. Louis, MO., U.S.A.), and suspended in 2507o inactivated newborn calf serum (Gibco Europe Ltd., Uxbridge, U.K.) absorbed with SE. The AET-treated SE were mixed with the mononuclear cells and incubated for 5 min at 37°C. The suspension was centrifuged at 200 g for 5 min and the cells incubated at 4°C overnight. The pellets were then resuspended and the number of rosettes counted. EA RFC. Fcy receptor (FcyR)-positive cells were also enumerated by a rosette assay (Hallberg, Gurner & Coombs, 1973). OE were incubated with 0.75 agglutinating units of rabbit IgG antibodies (A) and
washed and resuspended in PBS. One agglutinating unit is defined as the amount of the highest dilution of antiserum which agglutinates an equal amount of a 1070 suspension of OE. The indicator cells were mixed with the mononuclear cells treated with drug or solvent. The mixture stood at 4°C overnight, and the rosettes were then counted. E A C RFC. To demonstrate complement receptor (C3R)-positive cells, SE sensitized with 0.5 agglutinating units of rabbit IgM antibodies and human complement (C) were used (Matre & TOnder, 1976). The cells were suspended in barbital (Veronal)gelatine-buffered saline (VGB), then mixed with the mononuclear cells, incubated at 37°C for 30 min. The rosettes were then counted.
Phagocytizing cells The phagocytizing capacity of the cells incubated with drug or solvent was examined according to Wehinger & Hofacker (1976). Latex ®particles (Difco Lab., Detroit, MI., U.S.A.), 0.025 ml, were suspended in a mixture of 0.5 ml normal human plasma and 0.5 ml PBS. Equal volumes of the isolated mononuclear cells and the Latex suspension (0.2 ml of each) were mixed and incubated for 45 min at 37°C. After washing, the cells were resuspended in PBS and the number with ingested Latex particles were counted. Ig-positive cells Cells incubated with Latex particles were further incubated for 30 min at 4°C with 0.025 ml fluorescein-conjugated F(ab')2 fragments of IgG antibodies against human F(ab')2 fragments of Ig produced in goat (Nordic Immunological Lab., Tilburg, The Netherlands) (code No. 24-581, protein concentration 10 mg/ml, molar F / P ratio between 1 and 4). The cells were washed 3 times in PBS, mounted in PBS-buffered glycerol, and examined with a Leitz orthoplane microscope (Pernis, Forni & Amante, 1970). The percentage of lymhocytes with membranebound immunoglobulin (Ig), i.e. B-lymphocytes, was estimated by counting the total population of cells, and then those cells showing membrane fluorescence. The cells showing intracellular Latex particles were excluded from both counts. Cell viability. The viability of the cells treated with phenytoin, carbamazepine, propyleneglycol or PBS was determined by trypan blue dye exclusion, and was >90o70 in all experiments. Control experiments The percentage of cells in the various subpopulations were determined immediately after the separa-
The in vitro Effect of Phenytoin and Carbamazepine on Subpopulations of Human Blood Mononuclear Cells tion on Lymphoprep. The mononuclear cells were counted in a counting chamber before and after incubation with the drugs and solvents. The cell loss during the incubation procedure was less than 20°7o and did not differ with the different incubation media. Statistical evaluation
The percentage of mononuclear cells expressing the various cell markers after incubation with PBS was compared to the percentage after incubation with phenytoin, carbamazepine and propyleneglycol. A one-way analysis of variance was used. Subpopulations showing a statistical difference (P<0.05) in this assay were further examined for pair-wise differences between the percentage of cells after incubation with phenytoin, carbamazepine or propyleneglycol, using Scheff6's test (Winer, 1962). Values are given as mean -+ one standard deviation (SD).
RESULTS
Incubation with propyleneglycol did not affect the percentage of mononuclear cells in the various subpopulations compared to incubation with PBS, in any o f the experiments performed (Table 1). The percentage of ceils in the various subpopulations did not differ when counted immediately after the Lymphoprep separation and after incubation with PBS at 37°C for 30 min. The numbers of active E RFC and total E R F C (i.e. active and total T-lymphocytes) were significantly lower after incubation with the anti-epileptic drugs than after PBS incubation. The fractions of active and of total E RFC were both significantly lower after phenytoin incubation as compared to incubation with propyleneglycol alone. The differences between carbamazepine incubation and propyleneglycol incubation for active and for total E RFC were not statistically significant at a 5°7o level
Table 1. The effect of incubation with phenytoin 20 rag/1 (DPH), carbamazepine 10 mg/1 (CBZ), and propyleneglycol 1% (PG) compared to incubation with PBS, on the relative number of various mononuclear blood-cell subpopulations from 10 healthy blood donors Cell population
Active E RFC
Total E RFC
EA RFC
EAC RFC
Phagocytizing cells
lg-positive cells
Incubation medium
Percentage of cells (mean-+S.D.)
DPH CBZ PG PBS
17.6_+ 9.3 21.0_+10.9 25.9_+ 7.0 24.0_+ 6.1
P<0.05
DPH CBZ PG PBS
59.5_+15.6 60.3_+13.4 65.3_+15.8 65.3_+13.9
/:'<0.05
DPH CBZ PG PBS
19.6_+ 4.6 20.5_+ 5.1 20.7_+ 3.3 23.7_+ 2.5
P>O.05
DPH CBZ PG PBS
21.0+ 24.5_+ 23.6_+ 25.3_+
5.7 8.0 6.0 5.7
P>O.05
DPH CBZ PG PBS
11.6_+ 3.0 11.9_+ 4.2 12.9+_. 4.2 13.0_+ 3.1
P>O.05
DPH CBZ PG PBS
285
6.5_+ 5.0_+ 5.9_+ 5.9_+
2.4 2.5 2.8 2.0
Significance of difference within the relevant cell population
P>O.05
286
NILSERIK
Percentage of active E RFC
GILHUS
Percentage of active E RFC 30-
z,o
19
20 ¸
30
CBZ
20
~0
DPH
0
5 ~0 2'0 4'0 80 Drug conc.(mg/l)(Iog, scale)
10 i
o
Incubation Incubation Incubation with DPH with PG with CBZ
Fig. 1. A graphic comparison of the percentage of active E RFC after the incubation of mononuclear blood ceils with phenytoin 20 mg/l (DHP), carbamazepine 10 mg/l (CBZ) and propyleneglycol 1% (PG). Blood ceils were obtained from 10 healthy donors. (0.05
i
Fig. 2. The percentage of active E RFC after incubation with various concentrations of phenytoin (DPH) and carbamazepine (CBZ) in 1070propyleneglycol (mean values of incubation with blood ceils from 3 healthy donors). bound immunoglobulin were not significantly influenced by phenytoin and carbamazepine in concentrations similar to therapeutic serum concentrations in epileptic patients. However, the percentages of EA RFC, EAC RFC and phagocytizing cells were all slightly lower after phenytoin incubation (Table 1). Nevertheless, there was a considerable variability in the drug effect on ceils from the various donors, and in a few individuals mononuclear blood cell subpopulations other than active and total E RFC may have been suppressed by phenytoin. In one of the cell suspensions, there were 18% EA RFC after phenytoin incubation and 24°70 after propyleneglycol incubation; for EAC RFC there were 18 and 25°70, respectively; for phagocytizing cells 7 and 10070, respectively. Carbamazepine incubation did not affect the cell populations to the same extent in this cell suspension.
DISCUSSION
The results of this study clearly show that phenytoin affects some T-lymphocytes, so that their capacity to bind SE is reduced. The fraction of active E RFC (i.e. active T-lymphocytes) was significantly reduced. The reduction was relatively slight in most mononuclear blood cell suspensions, while in suspensions from some individuals the reduction was more marked, in agreement with previously reported data (Hornby & Mullen, 1977; Neilan, 1979). Also carbamazepine probably depresses the number of
The in vitro Effect of Phenytoin and Carbamazepine on Subpopulations of Human Blood Mononuclear Cells active T-lymphocytes, though not to the same extent as phenytoin does. The percentage of total T-lymphocytes was similarly reduced by phenytoin, and to a lesser extent by carbamazepine. Neilan (1979) found a slight, but significant reduction of total E RFC after incubation with phenytoin, while Hornby & Mullen (1977) concluded that phenytoin did not affect this lymphocyte subpopulation. In the latter study, the percentage after phenytoin incubation was compared to the percentage after incubation with the solvent, ethanol (0.25°7o). However, ethanol in this concentration reduces the total E RFC (Gilhus & Matre, 1982), and may thus mask the phenytoin effect. The results of the present study show that the reduction of active and total T-lymphocytes is dependent upon the concentrations of phenytoin and carbamazepine. For phenytoin, a reduction was present at concentrations in the lower end of the therapeutic range (10 rag/l), and was more marked using concentrations similar to those at the top of the therapeutic range (20 mg/1). For carbamazepine, the reduction was not present using low therapeutic concentrations (5 mg/l), and was only slight when high therapeutic concentrations (10 mg/1) were used. Phenytoin tended to reduce active T-lymphocytes more than it reduced total T-lymphocytes. Changes in the active rather than in the total T-cell population reflect certain pathological changes and cellular immune competence correlates better with the number of active T-cells than with the number of total T-cells (Fudenberg, Wybran & Robbins, 1975; Semenzato, Basso, Fagiolo, Pezzutto, Agostini, Cocito & Gasparotto, 1981). In in vitro experiments, phenytoin (20 mg/1) inhibits the DNA synthesis in mononuclear blood cells after phytohaemagglutinin (PHA) stimulation, and inhibits the mixed lymphocyte culture proliferation (MacKinney & Vyas, 1972; Sorrell & Forbes, 1975; Bluming, Homer & Khiroya, 1976). Also carbamazepine (10 rag/l) reduces P H A stimulated DNA synthesis (Sorrell & Forbes, 1975), thus showing that both these anti-epileptic drugs can alter T-cell function. In vivo, the interaction between phenytoin or carbamazepine and the immune system is more complex. Various tissues, cell populations and dissolved substances may be involved and the drugs as well as their metabolites are present. In a study examining active and total T-lymphocytes just before and after one day of phenytoin thereapy, no reduction was observed. The plasma concentration of phenytoin averaged 12.1 mg/l at the time of the second assay (Neilan & Leppik, 1980). Also, during long-term treatment, the relative numbers of active
287
and of total T-lymphocytes are probably unaffected by phenytoin or carbamazepine therapy (Seager, Jamison, Wilson, Hayward & Soothill, 1975; Massimo, Pasino, Rosanda-Vadali, Tonini, De Negfi & Saccomani, 1976; Gilhus et al., 1982a), though in one study 10 of 21 IgA-deficient patients on phenytoin had reduced numbers of T-lymphocytes (Fontana, Joller, Skvaril & Grob, 1978). The response of T-lymphocytes to mitogens, measured as synthesizing capacity, is probably moderately reduced in patients on phenytoin or carbamazepine (Sorrell & Forbes, 1975; Shakir, Behan, Dick & Lambie, 1978; Gilhus et al., 1982a). The B-lymphocytes, as examined in the present study, were not affected by the phenytoin or carbamazepine incubation. B-lymphocytes have membrane-bound immunoglobulin, and have FcyR and C3R. None of these mononuclear cell populations were reduced by the drugs. The percentage of B-lymphocytes is normal in peripheral blood from patients on phenytoin (Fontana et al., 1978; Gilhus et al., 1982a). However, mice injected with phenytoin have a reduction of plaque-forming cells in the spleen, and this reduction is probably due to a selective reduction of the B-cells in the spleen (Levo, Markowitz & Trainin, 1975; Queiroz & MuUen, 1980). The numbers of cells with phagocytizing capacity were not reduced by phenytoin or carbamazepine. The clinical consequences of the reduction of active and total T-lymphocytes, as demonstrated in the present study, are not clear. However, it is tempting to assume that the marked reductions seen in some of the incubation experiments may also occur in vivo, and that reduction of T-lymphocytes may contribute to the reduced immunological function and the increased frequency of infections in patients treated with anti-epileptic drugs (Shakir et al., 1978; Gilhus & Aarli, 1981). From the results of the present study, it is recommended that phenytoin and carbamazepine should be administered in the lowest effective dose, since the reduction in the number of T-lymphocytes is concentration-dependent within the therapeutic range for both drugs.
Acknowledgements--N. E. Gilhus is a Research Fellow of the Norwegian Cancer Society, Landsforeningen mot Kreft.
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NILS ERIKGILHUS REFERENCES
AARLI, J. A. (1976). Drug-induced IgA deficiency in epileptic patients. Archs Neurol., Chicago, 33,296-299. BLUMING, A., HOMER, S. & KHIROYA, R. (1976). Selective diphenylhydantoin-induced suppression of lymphocyte reactivity in vitro. J. Lab. clin. Med., 88,417-422. BREDESEN, J. E. & JOHANNESSEN, S. I. (1974). Simultaneous determination of some antiepileptic drugs by gas-liquid chromatography. Epilepsia (Boston), 15, 611-617. BOYUM, A. (1968). Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab. Invest., 21, (Suppl. 97). FONTANA, A., JOLLER, H., SKVAR1L,F. & GROB, P. (1978). Immunological abnormalities and HLA antigen frequencies in IgA deficient patients with epilepsy. J. Neurol. Neurosurg. Psychiat., 41,593-597. FUDENBERG, H. H., WYBRAN, J. & ROBBINS, D. (1975). T-rosette-forming cells, cellular immunity and cancer. New EngL J. Med., 292, 475-476. G1LHUS, N. E. & AARLI, J. A. (1981). Respiratory disease and nasal immunoglobulin concentrations in phenytointreated epileptic patients. Acta neuroL scand., 63, 34-43. GILHUS, N. E. & MATRE, R. (1982). In vitro effect of ethanol on subpopulations of human blood mononuclear cells. Int. Archs Allergy appl. Immun., 68, 382- 386. GILHUS, N. E., MATRE, R. & AARLI, J. A. (1982a). Lymphocyte subpopulations and lymphocyte function in phenytointreated patients with epilepsy. Int. J. Immunopharmac., 4, 43-48. GILHUS, N. E., STRANDJORD,R. E. & AARLI, J. A. (1982b). The effect of carbamazepine on serum immunoglobulin concentrations. A cta neurol, scand., 66, 172 - 179. HALLBERG, T., GURNER, B. W. & COOMBS, R. R. A. (1973). Opsonic adherence of sensitized ox red cells to human lymphocytes as measured by rosette formation. Int. ArchsAllergy appL Immun., 44, 500-513. HORNBY, C. & MULLEN, P. W. (1977). The effects of phenytoin on T-lymphocyte enumeration. Br. J. Pharmac. Chemother., 61,454. KAPLAN, M. E. 8£ CLARK, C. (1974). An improved rosetting assay for detection of human T lymphocytes. J. immun. Methods, 5,131-135. LEVO, Y., MARKOWITZ, O. & TRAININ, N. (1975). Hydantoin immunosuppression and carcinogenesis. Clin. exp. Immun., 19, 521-527. MACKINNEY, A. A., JR. & VYAS, R. (1972). Diphenylhydantoin-induced inhibition of nucleic acid synthesis in cultured human lymphocytes. Proc. Soc. exp. Biol. Med., 141, 89-92. MASSlMO, L., PASINO, M., ROSANDA-VADALI,C., TONIN1, G. P., DE NEGRI, M. & SACCOMANI,L. (1976). Immunological side-effects of anticonvulsants. Lancet, l, 860. MATRE, R. & TONDER, O. (1976). Complement receptors in human renal glomeruli. Scand. J. Immun., 5,437 441. NEILAN,B. A. (1979). Effect of phenytoin on T lymphocyte rosette formation. Curr. ther. Res., 25,557-563. NEILAN, B. A. & LEPPIK, I. E. (1980). Phenytoin and formation of T lymphocyte rosettes. Archs Neurol., Chicago, 37, 580-581. PERNIS, B., FORNI, L. & AMANTE, L. (1970). Immunoglobulin spots on the surface of rabbit lymphocytes. J. exp. Med., 132, 1001-1018. QUEIROZ, M. L. S. & MULLEN, P. W. (1980). The effects of phenytoin, 5-(para-hydroxyphenyl)-5-phenylhydantoin and valproic acid on humoral immunity in mice. Int. J. Immunopharmac., 2,224-225. SEAGER, J., JAMISON, D. L., WILSON, J., HAYWARD, A. R. & SOOTHILL,J. F. (1975). IgA deficiency, epilepsy, and phenytoin treatment. Lancet, 2,632-635. SEMENZATO,G., BASSO,G., FAGIOLO, U., PEZZUTTO,A., AGOSTINI, C., COCITO, M. G. & GASPAROTTO,G. (1981). Active and late rosette-forming cells: immunological and cytochemical characterization. Cell. Immun., 64,227-234. SHAKIR, R. A., BEHAN,P. O., DICK, H. & LAMBIE, D. G. (1978). Metabolism of immunoglobulin A, lymphocyte function, and histocompatibility antigens in patients on anticonvulsants. J. Neurol. Neurosurg. Psychiat., 41,307-311. SMITH,R., KERMAN,R., EZDINLI, E. & STEFANI, S. (1975). A modified assay for the detection of human adult active rosette forming lymphocytes. J. immun. Methods, 8,175 184. SORRELL, T. C. & FORBES, I. J. (1975). Depression of immune competence by phenytoin and carbamazepine. Studies in vivo and in vitro. Clin. exp. Immun., 20,273-285. WEH1NGER, H. & HOFACKER,M. (1976). Latex phagocytosis by polymorphonuclear leukocytes. In vitro and in vivo studies with a simple screening test. Eur. J. Pediat., 123, 125-132. WINER, B. J. (1962). Statisticalprinciples in experimentaldesign. McGraw Hill, New York.
0192-0561/83 $3,00 + ,00 @ 1983 International Society for Immunopharmacology.
THE I N VITRO EFFECT OF PHENYTOIN AND CARBAMAZEPINE ON SUBPOPULATIONS OF H U M A N BLOOD MONONUCLEAR CELLS NILS ERIK G1LHUS§ Department of Neurology and Broegelmann Research Laboratory for Microbiology, University of Bergen, Bergen, Norway
(Received 25 May 1982)
Abstraet--Mononuclear cells from peripheral blood of 10 healthy blood donors were incubated with phenytoin and carbamazepine dissolved in 1°70propyleneglycol. Phenytoin 20 mg/l significantly reduced the percentage of active E rosette-forming T-lymphocytes; to mean 17.6070compared to mean 25.9070 in control incubations (P<0.05). Carbamazepine 10 mg/1 reduced this percentage to 21.0070 (0.05
Both phenytoin and carbamazepine can induce a moderate reduction of the concentrations of IgA and IgM in serum, whereas deficiency of IgA occurs in about 5 070 of phenytoin-treated patients (Aarli, 1976; Gilhus, Strandjord & Aarli, 1982b). The total numbers of circulating lymphocytes are reduced in patients taking phenytoin, while the distribution between different lymphocyte subpopulations is unchanged (Gilhus, Matte & Aarli, 1982a). However, phenytoin probably reduces the number of T-lymphocytes in vitro (Hornby & MuUen, 1977; Neilan, 1979). Also carbamazepine may reduce the total number of lymphocytes (Sorrell & Forbes, 1975). The effect of this drug on lymphocyte subpopulations has not been examined. The synthesizing activity of mitogen-stimulated lymphocytes is reduced by both drugs (Sorrell & Forbes, 1975). In the present study, various subpopulations of mononuclear blood cells were enumerated before and after treating the cells with phenytoin and carbamazepine in concentrations similar to the serum concentrations in epileptic patients taking these drugs. In some experiments the effect of various drug concentrations was examined.
EXPERIMENTAL PROCEDURES
Peripheral blood mononuclear cells Heparinized peripheral blood was obtained from 10 consecutive healthy blood donors. Mononuclear cells were isolated on Lymphoprep ® (Nyegaard & Co., Oslo, Norway), washed 3 times in isotonic phosphate-buffered saline, pH 7.2 (PBS) and adjusted to a concentration of 3 × 106 cells/ml (BOyum, 1968).
Reagents Pure phenytoin powder (batch 427180) was provided by Nyegaard & Co., and pure carbamazepine powder (batch 002979) by Ciba-Geigy Pharma A/S, StrOmmen, Norway. The selected amount of each drug was dissolved in 1°70 propyleneglycol in PBS (v/v). Before incubation, the concentrations of phenytoin and carbamazepine were controlled by a gas-chromatographic method measuring the underivatized drug (Bredesen & Johannessen, 1974). Sheep and ox blood were collected in an equal volume of Alsever's solution. Before use the erythrocytes were washed 3 times in 10 volumes of PBS and
§ Correspondence to: Dr. N. E. Gilhus, Department of Neurology, N-5016 Haukeland Sykehus, Bergen, Norway. 283
284
NILS ERIK GILHUS
packed at 1000g for l0 min (100o70).
Incubation with phenytoin, carbamazepine or solvent One millilitre of the suspension of peripheral blood mononuclear cells was centrifuged at 200 g for 5 min, and the supernatant pipetted off. The cells were then resuspended in 0.1 ml of the various incubation media: phenytoin 20 mg/1, carbamazepine 10 mg/1, 107o propyleneglycol, and PBS. Cells from 3 donors were incubated with phenytoin and with carbamazepine in concentrations of 5, 10, 20, 40 and 80 mg/1. After incubation for 30 min at 37°C the cells were washed once in PBS, and the concentration of cells was adjusted to 3 x 106/ml. The mononuclear cells were incubated with the drugs or solvents within 4 h after the blood was drawn from the donor. Rosette techniques Sheep erythrocytes (SE) and ox erythrocytes (OE) were treated as indicated below, and made up to a 1°7o suspension. Equal volumes of the suspensions of indicator cells and of the mononuclear cells (0.05 ml of each) were mixed and incubated at the time and temperature indicated below. The percentage of mononuclear cells rosetting with the indicator cells (rosette-forming cells--RFC) were estimated by counting at least 200 cells in a counting chamber. A rosetting cell was defined as a cell binding 3 or more erythrocytes. A c t i v e E RFC. Active T-lymphocytes were enumerated by mixing the isolated mononuclear cells with the untreated SE. The cells were immediately centrifuged for 5 min at 200 g, the pellets gently resuspended, and the number of rosettes counted (Smith, Kerman, Ezdinli & Stefani, 1975). Total E RFC. The total number of lymphocytes with receptors for SE (i.e. total T-lymphocytes) were enumerated according to Kaplan & Clark (1974). Sheep erythrocytes were treated with 2-aminoethyli s o t h i o u r o n i u m bromide hydrobromide (AET) (Sigma Chemical Co., St. Louis, MO., U.S.A.), and suspended in 2507o inactivated newborn calf serum (Gibco Europe Ltd., Uxbridge, U.K.) absorbed with SE. The AET-treated SE were mixed with the mononuclear cells and incubated for 5 min at 37°C. The suspension was centrifuged at 200 g for 5 min and the cells incubated at 4°C overnight. The pellets were then resuspended and the number of rosettes counted. EA RFC. Fcy receptor (FcyR)-positive cells were also enumerated by a rosette assay (Hallberg, Gurner & Coombs, 1973). OE were incubated with 0.75 agglutinating units of rabbit IgG antibodies (A) and
washed and resuspended in PBS. One agglutinating unit is defined as the amount of the highest dilution of antiserum which agglutinates an equal amount of a 1070 suspension of OE. The indicator cells were mixed with the mononuclear cells treated with drug or solvent. The mixture stood at 4°C overnight, and the rosettes were then counted. E A C RFC. To demonstrate complement receptor (C3R)-positive cells, SE sensitized with 0.5 agglutinating units of rabbit IgM antibodies and human complement (C) were used (Matre & TOnder, 1976). The cells were suspended in barbital (Veronal)gelatine-buffered saline (VGB), then mixed with the mononuclear cells, incubated at 37°C for 30 min. The rosettes were then counted.
Phagocytizing cells The phagocytizing capacity of the cells incubated with drug or solvent was examined according to Wehinger & Hofacker (1976). Latex ®particles (Difco Lab., Detroit, MI., U.S.A.), 0.025 ml, were suspended in a mixture of 0.5 ml normal human plasma and 0.5 ml PBS. Equal volumes of the isolated mononuclear cells and the Latex suspension (0.2 ml of each) were mixed and incubated for 45 min at 37°C. After washing, the cells were resuspended in PBS and the number with ingested Latex particles were counted. Ig-positive cells Cells incubated with Latex particles were further incubated for 30 min at 4°C with 0.025 ml fluorescein-conjugated F(ab')2 fragments of IgG antibodies against human F(ab')2 fragments of Ig produced in goat (Nordic Immunological Lab., Tilburg, The Netherlands) (code No. 24-581, protein concentration 10 mg/ml, molar F / P ratio between 1 and 4). The cells were washed 3 times in PBS, mounted in PBS-buffered glycerol, and examined with a Leitz orthoplane microscope (Pernis, Forni & Amante, 1970). The percentage of lymhocytes with membranebound immunoglobulin (Ig), i.e. B-lymphocytes, was estimated by counting the total population of cells, and then those cells showing membrane fluorescence. The cells showing intracellular Latex particles were excluded from both counts. Cell viability. The viability of the cells treated with phenytoin, carbamazepine, propyleneglycol or PBS was determined by trypan blue dye exclusion, and was >90o70 in all experiments. Control experiments The percentage of cells in the various subpopulations were determined immediately after the separa-
The in vitro Effect of Phenytoin and Carbamazepine on Subpopulations of Human Blood Mononuclear Cells tion on Lymphoprep. The mononuclear cells were counted in a counting chamber before and after incubation with the drugs and solvents. The cell loss during the incubation procedure was less than 20°7o and did not differ with the different incubation media. Statistical evaluation
The percentage of mononuclear cells expressing the various cell markers after incubation with PBS was compared to the percentage after incubation with phenytoin, carbamazepine and propyleneglycol. A one-way analysis of variance was used. Subpopulations showing a statistical difference (P<0.05) in this assay were further examined for pair-wise differences between the percentage of cells after incubation with phenytoin, carbamazepine or propyleneglycol, using Scheff6's test (Winer, 1962). Values are given as mean -+ one standard deviation (SD).
RESULTS
Incubation with propyleneglycol did not affect the percentage of mononuclear cells in the various subpopulations compared to incubation with PBS, in any o f the experiments performed (Table 1). The percentage of ceils in the various subpopulations did not differ when counted immediately after the Lymphoprep separation and after incubation with PBS at 37°C for 30 min. The numbers of active E RFC and total E R F C (i.e. active and total T-lymphocytes) were significantly lower after incubation with the anti-epileptic drugs than after PBS incubation. The fractions of active and of total E RFC were both significantly lower after phenytoin incubation as compared to incubation with propyleneglycol alone. The differences between carbamazepine incubation and propyleneglycol incubation for active and for total E RFC were not statistically significant at a 5°7o level
Table 1. The effect of incubation with phenytoin 20 rag/1 (DPH), carbamazepine 10 mg/1 (CBZ), and propyleneglycol 1% (PG) compared to incubation with PBS, on the relative number of various mononuclear blood-cell subpopulations from 10 healthy blood donors Cell population
Active E RFC
Total E RFC
EA RFC
EAC RFC
Phagocytizing cells
lg-positive cells
Incubation medium
Percentage of cells (mean-+S.D.)
DPH CBZ PG PBS
17.6_+ 9.3 21.0_+10.9 25.9_+ 7.0 24.0_+ 6.1
P<0.05
DPH CBZ PG PBS
59.5_+15.6 60.3_+13.4 65.3_+15.8 65.3_+13.9
/:'<0.05
DPH CBZ PG PBS
19.6_+ 4.6 20.5_+ 5.1 20.7_+ 3.3 23.7_+ 2.5
P>O.05
DPH CBZ PG PBS
21.0+ 24.5_+ 23.6_+ 25.3_+
5.7 8.0 6.0 5.7
P>O.05
DPH CBZ PG PBS
11.6_+ 3.0 11.9_+ 4.2 12.9+_. 4.2 13.0_+ 3.1
P>O.05
DPH CBZ PG PBS
285
6.5_+ 5.0_+ 5.9_+ 5.9_+
2.4 2.5 2.8 2.0
Significance of difference within the relevant cell population
P>O.05
286
NILSERIK
Percentage of active E RFC
GILHUS
Percentage of active E RFC 30-
z,o
19
20 ¸
30
CBZ
20
~0
DPH
0
5 ~0 2'0 4'0 80 Drug conc.(mg/l)(Iog, scale)
10 i
o
Incubation Incubation Incubation with DPH with PG with CBZ
Fig. 1. A graphic comparison of the percentage of active E RFC after the incubation of mononuclear blood ceils with phenytoin 20 mg/l (DHP), carbamazepine 10 mg/l (CBZ) and propyleneglycol 1% (PG). Blood ceils were obtained from 10 healthy donors. (0.05
i
Fig. 2. The percentage of active E RFC after incubation with various concentrations of phenytoin (DPH) and carbamazepine (CBZ) in 1070propyleneglycol (mean values of incubation with blood ceils from 3 healthy donors). bound immunoglobulin were not significantly influenced by phenytoin and carbamazepine in concentrations similar to therapeutic serum concentrations in epileptic patients. However, the percentages of EA RFC, EAC RFC and phagocytizing cells were all slightly lower after phenytoin incubation (Table 1). Nevertheless, there was a considerable variability in the drug effect on ceils from the various donors, and in a few individuals mononuclear blood cell subpopulations other than active and total E RFC may have been suppressed by phenytoin. In one of the cell suspensions, there were 18% EA RFC after phenytoin incubation and 24°70 after propyleneglycol incubation; for EAC RFC there were 18 and 25°70, respectively; for phagocytizing cells 7 and 10070, respectively. Carbamazepine incubation did not affect the cell populations to the same extent in this cell suspension.
DISCUSSION
The results of this study clearly show that phenytoin affects some T-lymphocytes, so that their capacity to bind SE is reduced. The fraction of active E RFC (i.e. active T-lymphocytes) was significantly reduced. The reduction was relatively slight in most mononuclear blood cell suspensions, while in suspensions from some individuals the reduction was more marked, in agreement with previously reported data (Hornby & Mullen, 1977; Neilan, 1979). Also carbamazepine probably depresses the number of
The in vitro Effect of Phenytoin and Carbamazepine on Subpopulations of Human Blood Mononuclear Cells active T-lymphocytes, though not to the same extent as phenytoin does. The percentage of total T-lymphocytes was similarly reduced by phenytoin, and to a lesser extent by carbamazepine. Neilan (1979) found a slight, but significant reduction of total E RFC after incubation with phenytoin, while Hornby & Mullen (1977) concluded that phenytoin did not affect this lymphocyte subpopulation. In the latter study, the percentage after phenytoin incubation was compared to the percentage after incubation with the solvent, ethanol (0.25°7o). However, ethanol in this concentration reduces the total E RFC (Gilhus & Matre, 1982), and may thus mask the phenytoin effect. The results of the present study show that the reduction of active and total T-lymphocytes is dependent upon the concentrations of phenytoin and carbamazepine. For phenytoin, a reduction was present at concentrations in the lower end of the therapeutic range (10 rag/l), and was more marked using concentrations similar to those at the top of the therapeutic range (20 mg/1). For carbamazepine, the reduction was not present using low therapeutic concentrations (5 mg/l), and was only slight when high therapeutic concentrations (10 mg/1) were used. Phenytoin tended to reduce active T-lymphocytes more than it reduced total T-lymphocytes. Changes in the active rather than in the total T-cell population reflect certain pathological changes and cellular immune competence correlates better with the number of active T-cells than with the number of total T-cells (Fudenberg, Wybran & Robbins, 1975; Semenzato, Basso, Fagiolo, Pezzutto, Agostini, Cocito & Gasparotto, 1981). In in vitro experiments, phenytoin (20 mg/1) inhibits the DNA synthesis in mononuclear blood cells after phytohaemagglutinin (PHA) stimulation, and inhibits the mixed lymphocyte culture proliferation (MacKinney & Vyas, 1972; Sorrell & Forbes, 1975; Bluming, Homer & Khiroya, 1976). Also carbamazepine (10 rag/l) reduces P H A stimulated DNA synthesis (Sorrell & Forbes, 1975), thus showing that both these anti-epileptic drugs can alter T-cell function. In vivo, the interaction between phenytoin or carbamazepine and the immune system is more complex. Various tissues, cell populations and dissolved substances may be involved and the drugs as well as their metabolites are present. In a study examining active and total T-lymphocytes just before and after one day of phenytoin thereapy, no reduction was observed. The plasma concentration of phenytoin averaged 12.1 mg/l at the time of the second assay (Neilan & Leppik, 1980). Also, during long-term treatment, the relative numbers of active
287
and of total T-lymphocytes are probably unaffected by phenytoin or carbamazepine therapy (Seager, Jamison, Wilson, Hayward & Soothill, 1975; Massimo, Pasino, Rosanda-Vadali, Tonini, De Negfi & Saccomani, 1976; Gilhus et al., 1982a), though in one study 10 of 21 IgA-deficient patients on phenytoin had reduced numbers of T-lymphocytes (Fontana, Joller, Skvaril & Grob, 1978). The response of T-lymphocytes to mitogens, measured as synthesizing capacity, is probably moderately reduced in patients on phenytoin or carbamazepine (Sorrell & Forbes, 1975; Shakir, Behan, Dick & Lambie, 1978; Gilhus et al., 1982a). The B-lymphocytes, as examined in the present study, were not affected by the phenytoin or carbamazepine incubation. B-lymphocytes have membrane-bound immunoglobulin, and have FcyR and C3R. None of these mononuclear cell populations were reduced by the drugs. The percentage of B-lymphocytes is normal in peripheral blood from patients on phenytoin (Fontana et al., 1978; Gilhus et al., 1982a). However, mice injected with phenytoin have a reduction of plaque-forming cells in the spleen, and this reduction is probably due to a selective reduction of the B-cells in the spleen (Levo, Markowitz & Trainin, 1975; Queiroz & MuUen, 1980). The numbers of cells with phagocytizing capacity were not reduced by phenytoin or carbamazepine. The clinical consequences of the reduction of active and total T-lymphocytes, as demonstrated in the present study, are not clear. However, it is tempting to assume that the marked reductions seen in some of the incubation experiments may also occur in vivo, and that reduction of T-lymphocytes may contribute to the reduced immunological function and the increased frequency of infections in patients treated with anti-epileptic drugs (Shakir et al., 1978; Gilhus & Aarli, 1981). From the results of the present study, it is recommended that phenytoin and carbamazepine should be administered in the lowest effective dose, since the reduction in the number of T-lymphocytes is concentration-dependent within the therapeutic range for both drugs.
Acknowledgements--N. E. Gilhus is a Research Fellow of the Norwegian Cancer Society, Landsforeningen mot Kreft.
288
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