Clin. exp. Immunol. (1979) 38, 370-375.
The effects of azathioprine and levamisole on lymphocyte stimulation LUDMILA IVANYI Department of Oral Immunology and Microbiology, Guy's Hospital Dental School, London
(Accepted for publication 18 May 1979)
Azathioprine at 10 jug per ml caused approximately a 5000 inhibition of phytohaemagglutinin (PHA), purified protein derivative (PPD) or Veillonella induced in vitro lymphocyte stimulation. This suppression was significantly reduced in lymphocyte cultures from patients undergoing an eight week course of levamisole therapy. Azathioprine-induced suppression was also reversed when culturing lymphocytes in the presence of levamisole. On the other hand, levamisole in vitro failed to reverse the azathioprine suppression of cord blood lymphocyte responses. INTRODUCTION Levamisole acts as an immunostimulant, because it can restore or amplify delayed skin hypersensitivity (Tripodi, Parks & Brugmans, 1973) and in vitro cellular immunity in man (Lichtenfeld et aL, 1974; Lieberman & Hsu, 1976). It has also been reported that levamisole restored E-rosette formation of peripheral blood lymphocytes from healthy subjects after in vitro inhibition with azathioprine (Verhaegen et al., 1975). We have shown recently that treatment of patients with gingival inflammation with levamisole significantly enhanced the stimulation of lymphocytes by antigens from oral bacteria (Ivanyi & Lehner, 1977b). The aims of this study were to investigate the in vivo and in vitro effects of levamisole and azathioprine on in vitro lymphocyte stimulation by Veillonella antigen.
MATERIALS AND METHODS Patients. A group of five patients (two males and three females) between the ages of 20-40 years with gingival or periodontal disease was investigated. All subjects were well except for recurrent oral ulceration, for which they were receiving levamisole. The therapy consisted of administration of levamisole tablets, 300 mg per week for 8 weeks. During the last week of therapy, blood samples were taken. A control group of eleven patients with gingival or periodontal disease without levamisole therapy was matched for age (20-41 years) and sex (five males and six females). Human foetal cord blood was obtained immediately post-partum in a heparinized tube, care being taken to exclude contamination by maternal blood. Materials and treatment. Veillonella alcalescens sonicate was prepared as previously described (Ivanyi & Lehner, 1970) and used at an optimal concentration of 20 pg of protein per ml of culture (Ivanyi & Lehner, 1977b). Preservative-free PPD was used at a concentration of 10 pg per ml of culture, and PHA (Wellcome Reagents) was used at a dilution of 1:1000 per ml of culture. Levamisole (Janssen, Pharmaceutica, Beerse, Belgium) and azathioprine (Wellcome Reagents) were dissolved in saline and sterilized by passing through 0-22 u Millex TM filter. Azathioprine was used at concentrations varying from 1-50 pig per ml of lymphocyte cultures. As shown in Table 1, DNA synthesis of lymphocytes induced by various stimulants was inhibited by 50% in the presence of 10 pg of azathioprine and this concentration was used in all experiments. The degree of suppression was calculated from the formula:
of cultures with azathioprine and 100 cpm of cultures with stimulant alone Present address: Department of Oral Medicine, Institute of Dental Surgery, Eastman Dental Hospital, London. 0099-9104/79/1100-0370$02.00 C) 1979 Blackwell Scientific Publications % suppression
Azathioprine and levamisole effects on lymphocytes
TABLE 1. The effect of azathioprine on lymphocyte proliferation by various stimulants
Azathioprine ug/ml 10
PHA PPD Veillonella Saline
75,208+ 15,492 3966+ 698 2528+244 632+ 26-8
65,896± 13,433 3606+ 533 2236+208 521+ 16-7
12 10 12 3
42,681+4159 1957+ 480 1183+ 165 484+ 15-3
44 51 53 10
23,619+ 1336 804+ 89 596+81 341+ 12-0
69 80 77 36
* Mean values from three experiments. cpm = Counts per minute.
Lymphoid cell suspensions and DNA synthesis. Peripheral blood lymphocytes and foetal cord lymphocytes were isolated from heparinized blood by density gradient centrifugation on Ficoll-Triosil (Ivanyi & Lehner, 1977a). Lymphocytes were cultured at a concentration of 1 x 106 cells per ml of culture in medium RPMI 1640 enriched with L-glutamine (2 mmol/ml) penicillin (100 u/ml), streptomycin (100 ug/ml) and 10% autologous serum (peripheral blood lymphocytes) or 10% human AB serum (cord blood lymphocytes) (Ivanyi & Lehner, 1977a). The cultures were maintained and harvested as described previously (Ivanyi & Lehner, 1970). The results were expressed as counts per minute (cpm) per 1 x 106 viable lymphocytes.
RESULTS The effect of levamisole therapy on azathioprine sensitivity of lymphocyte stimulation Peripheral blood lymphocytes from five patients with periodontal disease were cultured during the last week of 2 months therapy with levamisole (Table 2). Eleven patients without levamisole therapy were used as controls. Lymphocytes from control patients showed marked inhibition of 14C-thymidine uptake in the presence of 10 pg/ml azathioprine; 42% suppression was observed in cultures with PHA, 56% suppression in cultures with PPD and 58% suppression in cultures with Veillonella. In contrast, only slight inhibition of DNA synthesis in lymphocyte cultures from patients undergoing treatment with levamisole was observed in the presence of azathioprine; 16% suppression in cultures with PHA and Veillonella and 13% suppression in cultures with PPD. Thus, the degree of azathioprine-mediated inhibition of lymphocyte responses to all three stimulants was significantly reduced in patients on levamisole therapy. TABLE 2. The effect of levamisole therapy on azathioprine sensitivity of lymphocyte stimulation
Controls without levamisole therapy (11 patients) 10
Stimulants PHA PPD Veillonella Saline
Levamisole therapy 300 mg/week for 8 weeks (5 patients)
66,101+5474 38,110+3708 3,297+ 809 1,468+ 291 2,132+ 126 684+ 51 559+ 34 502+ 23
Percentage inhibition 42 56 58 11
10 pg/ml azathioprine cpm
86,088+ 10.900 72,724+7918 3,240+ 219 3,716+ 406 2,588+ 207 2,163+ 285 601+ 77 557+ 48
Percentage inhibition 16 13 16 8
372 3' _
Levamisole Ag/mt FIG. 1. The effect of levamisole and azathioprine on lymphocyte stimulation induced by Veillonella. a = cpm of cultures with Veillonella. b = cpm of cultures with Veillonella and azathioprine (10 pg/ml). c = cpm of cultures with Veillonella, azathioprine and levamisole. n = 8.
The effect ofaddition oflevamisole to lymphocyte cultures The effect of in vitro added levamisole on the azathioprine sensitivity of lymphocyte responses was studied. Levamisole was added at the onset of cultures together with azathioprine and Veillonella. The maximal resistance to azathioprine suppression was found in cultures with levamisole at a concentration of 1 pg/ml (Fig. 1). The results presented in Table 3 show that levamisole alone had no effect on lymphocyte stimulation induced by Veillonella antigen (positive controls). Addition of levamisole or azathioprine alone also had no effect on unstimulated cultures (negative controls). Addition of azathioprine reduced lymphocyte stimulation by Veillonella by 57%0. However, the azathioprine-mediated inhibition of Veillonella-induced lymphocyte response was reversed in the presence of levamisole. The degree of levamisolemediated resistance to azathioprine suppression varied in individual subjects, from almost complete to only partial restoration of lymphocyte stimulation (Table 4). Furthermore, we examined the levamisole-induced resistance to azathioprine suppression of cord blood lymphocyte responses (Table 5). Cord blood lymphocytes showed marked inhibition of stimulation by Veillonella antigen in the presence of azathioprine (mean suppression of 63 + 5 63%). However, the inhibitory effect of azathioprine could not be restored by the addition of jig levamisole (mean suppression = 56%±4.97, P>0 05) or by levamisole over a wide range of concentrations (results not presented). TABLE 3. The effect of in vitro added levamisole on azathioprine sensitivity of lymphocyte responses Constituents in culture Counts per minute
Veillonella Azathioprine Levamisole 1 pg 10 ,pg 20 pg +
+ + -
+ + +
2326 2337 892 37 1784 497 524 484
307 59 57 51
n = Mean values from eight experiments.
Azathioprine and levamisole effects on lymphocytes
TABLE 4. Levamisole-induced resistance to azathioprine suppression of lymphocyte stimulation in individual patients
Percentage inhibition Patient number
Azathioprine + levamisolet
1 2 3 4 5 6 7 8
52 59 75 51 69 39 51 57
11 45 37 15 29 5 24 0
100- cpm Veillonella+azathioprine x 100. Veillonella
Veillonella + azathioprine + levamisole. - cpm t 100 V Veillonella + emi levamisole
TABLE 5. Lack of levamisole-induced resistance to azathioprine suppression of cord blood lymphocyte stimulation
Constituents in culture Veillonella
+ + + +
Counts per minute
14,335 14,499 5441 6473 4676 4241 4336 4613
1403 1938 1038 919 357 552 283 313
+ - -+ +
63 56 i
Mean values from eight experiments.
DISCUSSION Azathioprine at a concentration of 10 jug per ml of culture inhibited by approximately 5000 DNA synthesis in lymphocytes stimulated by PHA, PPD and Veillonella. However, decreased azathioprinemediated suppression was observed in lymphocyte cultures from patients undergoing an eight week course of levamisole therapy. The effect on in vitro addition of levamisole to lymphocyte cultures was also studied. The results indicate that levamisole in vitro depressed the azathioprine sensitivity of lymphocyte stimulation, though to a lesser extent, than prolonged in vivo administration of the drug to patients. The mechanism of azathioprine-mediated immunosuppression is still unclear. It has been shown that this drug is rapidly metabolized in vivo to 6-mercaptopurine, which is then transformed to 6-thioinosinic acid, which inhibits several steps in the synthesis of adenine, guanine (Elion, 1967) and DNA (Malamud et al., 1972). Indeed there is increasing evidence that azathioprine inhibits those reactions which require
cell division (Folb & Trounce, 1971; Rollinghof, Schrader & Wagner, 1973; Brown et al., 1975). However, it has also been suggested that the mechanism of this drug is through a blockade of the receptor site on the surface of the T cells (Bach & Dardenne, 1971). Levamisole may modulate the reactivity of T lymphocytes by its effect on cyclic nucleotide metabolism. It has been reported that levamisole in vitro increases cyclic GMP levels in cultured mouse T-enriched lymphocytes (Hadden et al., 1975). It was also suggested that levamisole has to be metabolized in order to become active (Van Ginckel & Hoebke, 1976) or that its enhanced effect is mediated by a hormonelike product (Renoux et al., 1974). Furthermore, injection of levamisole has been shown to depress the azathioprine sensitivity of murine rosette-forming spleen cells (Van Ginckel & Hoebke, 1976). Similar effects have been reported for drugs interfering with cyclic nucleotide metabolism (Bach & Bach, 1973) and for thymic extracts (Dardenne & Bach, 1973). We have also shown that azathioprine caused a 63% inhibition of Veillonella-induced stimulation of cord lymphocytes. It was surprising to find that this suppressive effect of azathioprine on cord lymphocyte responses could not be restored by the addition of levamisole. The reason for this is unclear at present, but at least two mechanisms can be considered. Several authors have reported that immature T cells are more sensitive to inhibition by azathioprine. Bach, Dardenne & Fournier (1969) demonstrated a subpopulation of spontaneous rosette-forming cells in mice, which were sensitive to low concentrations of azathioprine. In the thymus, this drug was found to deplete a particular population of thymocytes without affecting the hydrocortisone resistant thymocytes (Poulter, Bradley & Turk, 1974). 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