Clin. exp. Immunol. (1978) 32, 411-418.
Effect of niridazole in cellular immunity in vivo and in vitro W. SOLBACH, H. WAGNER & M. ROLLINGHOFF Institut fur Medizinische Mikrobiologie der Johannes Gutenberg, Universitit Mainz, Mainz, West Germany
(Received 16 January 1978) SUMMARY
The influence of niridazole, an anti-helminthic drug, on cell-mediated immune responses was investigated. Allograft rejection in mice as well as the in vitro induction of cytotoxic T lymphocytes (CTL) against murine alloantigen were used as the test system. Repeated daily oral treatment of host mice with niridazole (100 mg/kg) prior to and during allotransplantation resulted in the postponement of graft rejection, inducing a transitory functional state of allograft tolerance. The time interval between the termination of niridazole administration and onset of graft rejection was estimated to be 5-7 days. In order to test the effect of niridazole or its derivatives on the in vitro induction of alloreactive CTL, the serum or urine of mice which were treated with niridazole were added to the cultures, instead of adding niridazole directly to the cultures. Such serum and urine were found to be inhibitory for in vitro induction of CTL. The serum and urine had no effect on the effector phase of CTL.
INTRODUCTION In recent studies, niridazole, an anti-helminthic drug used for 12 years in the treatment of schistosomiasis, has been shown to be a potent suppressant of T cell-mediated immune reactions in a number of species. Given orally to mice, niridazole markedly inhibited both granuloma formation around Schistosoma mansoni eggs and the delayed footpad swelling elicited by soluble schistosome egg antigen (Mahmoud & Warren, 1974). Niridazole also delayed the rejection of mouse and rat allografts (Mahmoud, Warren & Webster, 1975; Salaman et al., 1977). When administered to guinea-pigs sensitized to a modified bovine y-globulin, niridazole abolished the delayed cutaneous reactivity and blocked the production of migration inhibition factor (Daniels, Warren & David, 1975). In man, niridazole resulted in reduced or ablating skin reactions to tuberculin, mumps and schistosome antigens (Webster et al., 1975). Antibody responses, however, were only minimally affected by the drug (Pelley et al., 1975). During an investigation of the mechanism of the suppression of the T cell-mediated immune response, it was suggested that niridazole metabolites produced by niridazole administration might be responsible for this activity (Mahmoud et al., 1975; Webster et al., 1975). This hypothesis was supported by the observation that sera and urine, from animals treated with niridazole, blocked the in vitro production of migration inhibition factor, while niridazole itself was inactive (Daniels, Fajardo & David, 1975; Lucas et al., 1977). The studies reported here were undertaken to analyse further the niridazole-induced prolongation of skin allograft survival in mice, as well as to investigate the effect of sera and urine from mice treated with niridazole on the generation of cytotoxic T lymphocytes (CTL) induced in a mixed lymphocyte Correspondence: Dr M. Rollinghoff, Institut fur Medizinische Microbiologie der Universitat Mainz, Hochhaus am Augustusplatz, D-6500 Mainz, West Germany. 0099-9104178/0600-0411$02.00 (©1978 Blackwell Scientific Publications
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reaction. The data confirm the reversible effect of niridazole in delaying allograft rejection and indicate that the time interval between termination of drug administration and the beginning of graft rejection is about 6 days. Furthermore, the results indicate that sera and urine from niridazole-treated mice are capable of suppressing the in vitro induction of CTL, when given to cultures in their early phase. MATERIALS AND METHODS Mice. CBA/J and BALB/c inbred mice were purchased from GI Bomholtgaard, Ry, Denmark, and used at an age of 8-12 weeks. Niridazole. Niridazole [1-(5-nitro-2-thiazolyl)-2 imidazolodinone], a gift of Ciba-Geigy, Wehr/Baden, West Germany, was suspended in distilled water at a concentration of 5 0 mg/ml. The mice, anaesthetized with ether, received niridazole orally by a plastic tube inserted into the oesophagus, at a dose of about 100 mg/kg. Sera. Blood was obtained from mice by bleeding through the orbit and allowed to stand for 30 min at 4VC. Sera were centrifuged at 3000 g for 20 min. They were not inactivated, and they were diluted 1:10 in culture medium, sterilized through a Millipore filter (pore size 0-22 pm) and stored at - 20'C until used. Sera from mice which had received niridazole daily on at least 7 consecutive days were designated NIR sera. Urine. Urine was reliably obtained from mice by kneading the skin of the neck and at the same time carefully pulling of the tail, thereby triggering a micturition reflex. Urine was processed in a similar way to the serum. Urine from mice which had received niridazole daily on at least 4 consecutive days was designated NIR urine. Skin allografting. Skin grafting of BALB/c tail skin on CBA mice was done according to the method described by Billingham (1961). Tail skin was cut across to give grafts of 1.0 cm2 and these were applied to the dorso-lateral thorax of the recipients and covered with a non-adherent dressing and a bandage. Dressings were removed on day 9, and the grafts were considered rejected when two independent examiners scored more than 80%. of the graft as necrosed. In vitro induction of alloantigen-reactive cytotoxic T lymphocytes. The basic culture system has been described in detail (Rollinghoff et al., 1975; Wagner et al., 1976). In brief: spleen cells were used as source of responding and stimulating cells. Before culture, the stimulating cells received a dose of2000 rad (Philips machine, RT 200) at a dose rate of 320 rad/min. The cells were cultured in multi-dish culture trays (Costar 3524, Costar Cambridge, Massachusetts) using Dulbecco's modified Eagle's medium (DMEM) (Grand Island Biological Co., Grand Island, New York), supplemented with 5 x 10-5M 2-mercaptoethanol and 5% foetal calf serum. CBA responder cells (4 x 106) and BALB/c stimulator cells (106) were cultured in a volume of 2-0 ml in a humidified atmosphere of 10%4 CO2 for 5 days. Then the cytotoxic activity generated was tested in a 51Cr-release assay. To some cultures graded concentrations, of NIR serum or NIR urine were added in a final volume of 0-2 ml culture medium. Cytotoxic assay. The cytotoxic activity of the CTL induced in vitro was tested according to the method described previously (Wagner et al., 1976). In brief: a constant number of ' Cr-labelled target cells (2 x 104) was mixed with graded numbers of attacker CTL in a volume of 1-0 ml DMEM in round bottom plastic tubes (No. 175, Greiner, Nurtingen, West Germany). After 3 hr incubation, the radioactivity of both the supernatant and pellet was determined in a Packard autogamma counter. Percentage specific lysis was calculated according to the formula described elsewhere (Wagner et al., 1976). The standard deviation (s.d.) of triplicate assays was calculated. The data are presented without s.d. since in all experiments listed the s.d. of the percentage lysis was less than 5%. Statistical analysis was performed using the Student's t-test.
RESULTS
Influence of niridazole on murine allograft rejection The effect of niridazole on skin allograft survival was investigated in CBA mice, which had received a BALB/c strain-derived skin graft. The host mice were treated orally with 100 mg/kg niridazole (or with placebo) daily from day -3 to day 9, and were grafted on day 0. As a control, each experimental mouse also received a syngeneic CBA skin graft. The results are detailed in Table 1: the allografts survived for 18-3 + 1 0 days as compared to a mean survival of 10-5 + -14 days in placebo-treated controls. None of the syngeneic control grafts were rejected. The animal mortality was up to two out of a group of nineteen. These data confirm an earlier report by Mahmoud et al. (1975) and clearly demonstrate niridazole to be an immunosuppressive agent in prolonging skin allograft survival. To test whether niridazole may be of use as an immunosuppressant over a longer period of time, CBA mice received doses of 100 mg/kg of niridazole orally for graded periods of time. The drug treatment was started 2 days before transplanting the BALB/c skin grafts and continued until day 9, 12, 15, 18 or 24. Allograft rejection was delayed in all experimental groups (Fig. 1), and a correlation was found to
Immunosuppressive effect of niridazole
413
TABLE 1. Allograft survival in niridazole-treated mice Treatment with niridazole (100 mg/kg)
Origin of skin graft
Host
CBA
CBA CBA BALB/c BALB/c
(days)
Allograft survival time (days+ s.d.)
No. of mice
Mortality
Placebo control -3to9 Placebo control -3 to 9
> 30 >30 10-5+1-4 18-3+ 1-0
19 19 19 19
2/19 0/19 1/19 2/19
exist between the time of termination of niridazole administration (day 9, 12, 15, 18 and 24) and the timepoint at which 50%4 of the grafts were rejected (day 17, 19, 21, 23 and 30). The mean interval was estimated to be 6-20+ 1 50 days. Syngeneic CBA control skin grafts were accepted permanently over an observation period of 40 days. The mortality of the mice of the experimental groups and the controls did not differ; up to two mice per group died during the experiment, mostly due to the repetitive (up to twenty-six times) gastro-oesophagical drug application, which included ether anaesthesia. All surviving mice appeared healthy over the period of observation. These data suggested that the time of allograft rejection can be arbitrarily determined by predetermining the period of niridazole treatment. Effect of niridazole on the capacity of spleen cells to generate cytotoxic T cells in vitro A first set of experiments were designed to find out if spleen cells were altered in their capacity to generate CTL in vitro when the donors of the spleen cells were treated previously with niridazole. CBA mice received niridazole (100 mg/kg) orally on 7 consecutive days. Subsequently spleen cells (4 x 106) from these mice were cultured as responder cells together with 106 BALB/c strain-derived X-irradiated (2000 rad) stimulator cells for 5 days. The cytotoxic activity generated was measured by a 51Cr-release assay, the results of which are given in Table 2. In six independent experiments, niridazole treatment of the mice that had provided the responder cells over 1 to 11 days had not resulted in a significant decrease in the capacity of the spleen cells to generate CTL in vitro. Similarily, no effect was observed when the donor mice received the drug only once during the period from day -7 to -1 (data not shown). Since pre-treatment of donor mice with niridazole had little inhibiting effect on the capacity of spleen cells of such mice to generate alloreactive CTL, it was of interest to test whether niridazole was able to affect significantly the induction of CTL in vitro, when present during the 5 day culture period. Previous studies had suggested that it was not niridazole, which is insoluble in water, but metabolites ofniridazole that were responsible for the reported immunosuppressive activity (Daniels et al., 1975). Therefore, we 10080-
60 40-
1(I)~~~~~~I
020 0
10
12
14
16
18
20 22 24 26 Days after transplantation
28
30
32
34
FIG. 1. Time relationship between survival ofBALB/c skin grafts on CBA hosts and duration of oral niridazole
administration. (o) Group I (n = 40), without niridazole treatment; (o) group II (n = 25), niridazole daily
100 mg/kg from day -2 to 9; (U) group III (n = 6); niridazole from day -2 to 12; (L) group IV (n = 6), days -2 to 15; (A) group V (n = 6), days -2 to 18; and (A) group VI (n = 17), days -2 to 24. C
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TABLE 2. Effect of niridazole treatment of the donor mice on the capacity of donor mice-derived responder spleen cells to generate cytotoxic T cells in vitro Specific lysis (%)t of Experiment No.:
Pre-treatment of donor mice (CBA) of the responder spleen cells
NaCi (0 9%) on 11 consecutive days Niridazole (100 mg/kg) on 11 consecutive days§
In vitro CTL induction system*
1
2
3
4
5
6
Mean specific lysis (%)
CBA anti-BALB/c
70
68
96
72
97
101
84-0+
CBA anti-BALB/c
46
47
92
59
85
88
69-5+
* 4 x 106 responder spleen cells were cultured together with 106 X-irradiated (2000 rad) BALB/c stimulator cells for 5 days. t Effector to P815 target cell ratio was 30:1. The assay time of the 51Cr-release test was 3 hr. Background lysis of P815 was 11%. t These values did not differ significantly (P> 0 05). § As control, some of the CBA mice received BALB/c skin grafts, similar to group II in Fig. 1, and they all showed an enhanced graft survival.
used serum from mice treated daily for at least 7 consecutive days with doses of 100 mg/kg of niridazole as a source of niridazole metabolites (subsequently abbreviated as NIR serum) and tried to determine the effect of NIR serum on the induction of CTL in vitro. CBA responder cells (4 x 106) were cultured together with 106 X-irradiated (2000 rad) BALB/c stimulator cells in the presence of graded concentrations of NIR serum. As can be seen in Table 3, the presence of NIR-serum during the culture period markedly inhibited the in vitro generation of CTL. Since not only the serum but also the urine of animals treated with niridazole was shown to contain metabolites of the drug (Faigle & Keberle, 1969; Lucas et al., 1977), we also tested the effect of urine obtained from mice which had received the compound (100 mg/kg) daily by mouth for more than 4 days on the induction of CTL in vitro. On the basis of observations on the pharmacodynamics of niridazole (Faigle & Keberle, 1969), we assumed that after 4 days of daily treatment with niridazole a constant concentration of either niridazole metabolites or a niridazole-induced immunosuppressive factor would be reached in the urine. Such urine was designated NIR urine. The results of an experiment in which the effect of graded concentrations of NIR urine on the induction of CTL in vitro was compared with the effect of normal urine are given in Fig. 2. The data showed that NIR-urine inhibited the generation of CTL in vitro in the same way as NIR serum. TABLE 3. Effect of graded concentration of NIR serum on the generation of alloreactive cytotoxic T cells in vitro
Concentration of serum in the culture system (%)*
Specific lysis (%)
NIR serum
Normal mouse serum
50: It
5:1
0 1 2 3
3 2 1 0
99 42 8 6
40 7 2 4
* CBA anti-BALB/c CTL were induced as described in Table 2. t Effector to P815 target cell ratio. Background lysis was
9%.
Immunosuppressive effect of niridazole
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904
I0
U 21%
Co (n
Volume (%)
FIG. 2. Effect of graded concentrations of NIR urine on the generation of CBA anti-BALB/c cytotoxic T cells in vitro. 4x 106 CBA normal spleen cells were incubated together with 106 X-irradiated BALB/c-derived spleen cells. Culture medium contained graded concentrations of urine, either derived from normal mice (o) or niridazole pre-treated mice (0). Abscissa shows the lysis of P815 target cells. Effector:target cell 50:1. Background lysis was less than 11%. Exact values in parenthesis.
In the next experiment, we tried to determine whether the suppression of CTL induction by NIR serum and NIR urine was due to interference of the early phase (i.e. antigen recognition) or of the late phase (i.e. differentiation of pre-CTL into effective CTL) of the immune reaction by metabolites of niridazole. In order to test for this, NIR serum or NIR urine were added to the culture in vitro at various times after its onset. The final concentration of NIR serum and NIR urine in the cultures was 2% and 1% respectively. As can be seen in Fig. 3, the presence of metabolites of niridazole in the form of NIR serum or NIR urine during the whole culture period of 5 days markedly affected the induction of alloreactive CTL. If, however, the metabolites were present only during the late phase of the culture (day 80-r
60 1.
a
0 0
401-
c
:aTo
I
._
0 ._
20
C)
F i
L---L-
0
I 2
I .I I
I.I I
1. I.
I "-f
M&A
Jg;Z.;W6-
------
4 3 Day of addition of NIR serum or NIR urine to the culture
1
FIG. 3. Effect of adding 2% NIR serum (n) or 1% NIR urine (0) at various times after the onset of a mixed lymphocyte culture on the induction of CBA anti-BALB/c cytotoxicity. The ordinate represents the percentage inhibition of the cytotoxicity generated in a 5 day culture. The percentage inhibition was calculated by comparing the cytotoxicity induced in cultures supplemented with NIR serum (or urine) and normal serum (or urine). The effector:P815 target cell ratio was 50: 1. The cytotoxic activity of control cultures was 63%. Background lysis was 14%.
W. Solbach, H. Wagner & M. Rdi1inghoff
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TABLE 4. Effect of NIR serum and NIR urine on the cytolytic activity of CTL*
Concentration (%) of: Serum -
Specific lysis (%)t
Urine
Normal serum
-
68
NIR serum
0*5 0 75
65
65
66
77
71
77
1.0
1*5 2-0
30
Normal urine
NIR urine
65
67
62
64
58
57
* In vitro generated CBA anti-BALB/c CTL (106) were incubated at 37'C in 10 ml culture medium containing 1% foetal calf serum and CBA serum or CBA urine in the concentrations indicated. 2 hr later 2x 104 51Cr-labelled P815 target cells were added, the cell mixtures were briefly centrifuged (1 min, 1000 rev/min) and incubated for a further 3 hr.
t Background lysis was 12%.
3-5), they had only a moderate influence on the CTL generation. From these data it is concluded that the metabolites of niridazole appear to interfere with the early phase of the induction of CTL.
Effect of NIR serum and NIR urine on the effector phase of cytotoxic T lymphocytes The cytotoxic activities of a given population of CBA anti-BALB/c CTL was measured against P815 target cells in a 51Cr-release assay for 3 hr under standard conditions. A parallel experiment was set up in the presence of graded concentrations of NIR serum or NIR urine. No effect of NIR serum or NIR urine on the effector phase of CTL was observed, even in concentrations of 3Y% and 2% respectively (Table 4), indicating that metabolites of niridazole did not interfere with the killing mechanism of CTL, not were they toxic for CTL, when present for as long as 5 hr. DISCUSSION In the present study the effect of niridazole on skin allograft survival and the in vitro induction of alloantigen-reactive CTL was tested. Four points appear to be noteworthy. Firstly, enhanced graft survival times were observed after repeated daily doses of 100 mg/kg niridazole, administered by the oral route, thereby confirming earlier studies by Mahmoud et al. (1975) and Salaman et al. (1977). Secondly, kinetic experiments revealed that by selecting the length of time of niridazole treatment, the time of the prolongation of graft survival could be determined. Thus an interval of 5-7 days between the termination of drug treatment and graft rejection was observed, independent of the length of niridazole treatment. Thirdly, the generation of alloreactive CTL in vitro was found to be suppressed, provided serum or urine from mice treated orally with niridazole was present in the culture during the early phase of the immune reaction (day 1-3). On the other hand, both NIR serum and NIR urine had no effect on the cytolytic effector phase of CTL. In contrast to niridazole, other drugs, such as the alkylating agents, steroids, purine and pyrimidine analogues, as well as 6-mercaptopurine and azathioprine, have all failed to enhance the survival of murine skin allografts across the major histocompatibility barrier when administered in non-toxic doses (Santos, 1967, 1972; Schwartz, 1965; Stewart, 1969). Only repeated doses of anti-thymocyte serum and sublethal total body radiation have been described as prolonging graft survival (Mandel & Asofsky, 1968; Medawar, 1963). Therefore, it has been suggested that niridazole may be useful in clinical organ transplantation (Mahmoud & Warren, 1974). Indeed, the low incidence of major side effects in patients with
Immunosuppressive effect ofniridazole
417
schistosomiasis and guinea-worm infestation treated with niridazole (Lucas, Oduntan & Gilles, 1969), as well the ability to programme the prolongation of skin allograft survival by repeated daily oral treatment with the drug (Fig. 1), would seem to recommend niridazole as a potent immunosuppressant in organ transplantation. In keeping with this is the observation that niridazole, when tested in rats either alone or in combination with prednisolone and/or azathioprine, has a marked enhancing effect on the survival ofcardiac allografts. As no toxic effects of the drug have been noted (Salaman et al., 1977), the allograft survival-enhancing effect of niridazole should now be tested in larger animals, such as dogs and primates. Whereas the survival of allogeneic skin grafts was considerably prolonged in niridazole-treated mice, to our surprise spleen cells from such animals reacted almost normally in vitro. In response to allogeneic stimulator cells, spleen cells from such mice were almost invariably able to differentiate into CTL, when compared to control spleen cells (Table 2). A possible explanation for this could be that an in vivo effective niridazole-dependent suppressive activity is washed off during the preparation of the responder lymphocytes for the in vitro culture. A similar dissociation between in vivo and in vitro reactivity was reported by Daniels et al. (1975). These authors had observed that in guinea-pigs niridazole treatment abolished delayed cutaneous reactivity, whereas little or no effect was seen when cells from these animals were tested for migration inhibitory factor (MIF). The mechanism of niridazole-induced suppression of cell-mediated immune reactivity is also of interest. Mahmoud et al. (1975) and Webster et al. (1975) were the first to suggest that it was not niridazole itself but metabolites of niridazole that were responsible for the immunosuppressive effect. They had shown that sera taken from niridazole-treated animals markedly diminished antigen-induced inhibition of sensitized peritoneal exudate cells in vitro, although niridazole itself was inactive. As described before, NIR serum blocked the MIF production but not the MIF action on macrophages. The effect of NIR serum was found to be reversible, in that sensitized lymph node cells, incubated for 24 hr with NIR serum, then washed and re-incubated with antigen in normal serum, produced normal amounts of MIF (Daniels et al., 1975). Recently, Lucas et al. (1977) have reported that the immunosuppressive activity of niridazole can also be detected in the urine of rats and patients receiving niridazole. We now present evidence that NIR serum as well as NIR urine is capable of diminishing the in vitro induction of alloantigen-reactive CTL (Fig. 2 and Table 3). It is interesting to note that the suppression became effective only when NIR serum or NIR urine was present during the first 2-3 days of culture (Fig, 3). Addition to the culture at day 4 had no significant effect on the in vitro induction of alloreactive CTL, nor was there any effect on the capacity of the CTL to lyse appropriate target cells (Table 4). Similarly, Jones et al. (1977) have recently reported that NIR sera and NIR urine dialysates were effective in inhibiting the human mixed lymphocyte reaction, when added before or within 30 min of the initiation of the reaction. From these data, as well as from unpublished data of Vadas, Bernard & Whitelaw (personal communication), who studied the effect of niridazole on delayed-type hypersensitivity, it may be deduced that niridazole acts on cell-mediated immune reactions via its metabolites during the early phase of T-cell sensitization, but not during the late (effector) phase. Whether niridazole metabolites interfere at the level of antigen presentation, or directly at the level of antigen-reactive T cells, remains an open question. What is the nature of the observed immunosuppressive activity? As already stated, niridazole appears to react not by itself, but only by metabolites (Daniels et al., 1975; Lucas et al., 1977). This is in good agreement with earlier data, which have indicated that niridazole, after administration to an animal, is converted rapidly to unknown metabolites, which have a longer half-life than niridazole, presumably because the drug metabolites bind to plasma proteins (Faigle & Keberle, 1969). Daniels et al. (1975) have also shown that the immunosuppressive activity of NIR serum can be removed by dialysis. However, despite the fact that Lucas et al. (1977) succeeded in enriching the suppressive activity 100- to 1000-fold by combined solvent extraction and chromatographic techniques, the substance responsible for the suppressive activity has not, as yet, been chemically identified. This work was supported by Sonderforschungsbereich 107 (Mainz) of the Deutsche Forschungsgemeinschaft. We are grateful to Mrs L. Ptschelinzew and Mr C. Hardt for their expert technical help.
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REFERENCES BILLINGHAM, R.E. & MEDAWAR, P.B. (1951) The technique vs-host assay as a measure of homograft reactivity. 1. of free skin grafting in mammals. J. exp. Biol. 28, 385. Immunol. 100, 1319. DANIELS, J.C., FAJARDO, I. & DAVID, J.R. (1975) Two stages MEDAWAR, P.B. (1963) The use of antigenic tissue extracts in lymphocyte mediator production by differential susceptibility to blockade using niridazole. Proc. nat. Acad. Sci. (Wash.), 72, 4569. DANIELS, J.C., WARREN, K.S. & DAVID, J.R. (1975) Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compound niridazole. 7. Immunol. 115, 1414. FAIGLE, J.E. & KEBERLE, H. (1969) Metabolism of niridazole in various species, including man. Ann. N. Y. Acad. Sci. 160, 544. JONES, B.M., BIRD, M., HOWELLS, M., MASSEY, P.R., MILLAR, D., MILLER, J.J., REEVES, S. & SALAMAN, J.R.
(1977) Inhibition of human mixed lymphocyte reactions by sera and urine dialysates from niridazole-treated rats. Transplantation, 24, 134. LUCAS, S.V., DANIELS, J.C., SCHUBERT, R.D., SIMPSON, J.M., MAHMOUD, A.A.F., WARREN, K.S., DAVID, J.R. & WEBSTER, L.T. (1977) Identification and purification of immunosuppressive activity in the urine or rats and a human patient treated with niridazole. J. Immunol. 118, 418. LUCAS, A.E., ODUNTAN, S.O. & GILLES, H.M. (1969) Niridazole in guinea-worm infection. Ann. N. Y. Acad. Sci. 160, 729. MAHMOUD, A.A.F. & WARREN, K.S. (1974) Anti-inflammatory effects of tartar emetic and niridazole- suppression of schistosoma egg granuloma. J. Immunol. 112, 222. MAHMOUD, A.A.F., WARREN, K.S. & WEBSTER, L.T. (1975) Niridazole. II. A potent long-acting suppressant of cellular hypersensitivity. J. Immunol. 114, 279. MANDEL, M.A. & ASOFSKY, R. (1968) The effects of heterologous anti-thymocyte sera in mice. I. The use of a graft-
to weaken the immunological reaction against skin homografts in mice. Transplantation, 1, 21. PELLEY, R.P., PELLEY, R.J., STAVITSKY, A.B., MAAHMOUD, A.A.F. & WARREN, K.S. (1975) Niridazole, a potent longacting suppressant of cellular hypersensitivity. III. Minimal suppression of antibody responses. .. Immunol. 115, 1477. ROLLINGHOFF, M., PFIZENMAIER, K., TROSTMANN, H. & WAGNER, H. (1975) T-cell proliferation in mixed lymphocyte culture does not necessarily result in the generation of cytotoxic T cells. Europ... Immunol. 5, 560. SALAMAN, J.R., BIRD, M., GODFREY, A.M., JONES, B., MILLAR, D. & MILLER, J. (1977) Prolonged allograft survival with niridazole, azathioprine, and prednisolone. Transplantation, 23, 29. SANTOS, G.W. (1967) Immunosuppressive drugs. Fed. Proc. 26, 906. SANTOS, G.W. (1972) Chemical immunosuppression, Transplantation (eds J.J. Najarian and R.L. Simmons). p. 206. Lea and Febinger, Philadelphia. SCHWARTZ, R.S. (1965) Immunosuppressive drugs. Prog. Allergy, 9, 246. STEWART, P.B. (1969) Failure of 6-mercaptopurine to prolong the survival of skin allografts in mice. Transplantation, 7, 498. WAGNER, H., STARZINSKI-POWITZ, A., PFIZENMAIER, K. & ROLLINGHOFF, M. (1976) Regulation of T-cell-mediated cytotoxic allograft responses. I. Evidence for antigenicspecific suppressor T-cells. Europ. J. Immunol. 6, 873. WEBSTER, L.T., BUTTERWORTH, A.F., MAHMOUD, A.A.F., MNGOLA, E.N. & WARREN, K.S. (1975) Suppression of delayed hypersensitivity in schistosome-infected patients by niridazole. New Engl. . Med. 292, 1144.
Effect of niridazole in cellular immunity in vivo and in vitro W. SOLBACH, H. WAGNER & M. ROLLINGHOFF Institut fur Medizinische Mikrobiologie der Johannes Gutenberg, Universitit Mainz, Mainz, West Germany
(Received 16 January 1978) SUMMARY
The influence of niridazole, an anti-helminthic drug, on cell-mediated immune responses was investigated. Allograft rejection in mice as well as the in vitro induction of cytotoxic T lymphocytes (CTL) against murine alloantigen were used as the test system. Repeated daily oral treatment of host mice with niridazole (100 mg/kg) prior to and during allotransplantation resulted in the postponement of graft rejection, inducing a transitory functional state of allograft tolerance. The time interval between the termination of niridazole administration and onset of graft rejection was estimated to be 5-7 days. In order to test the effect of niridazole or its derivatives on the in vitro induction of alloreactive CTL, the serum or urine of mice which were treated with niridazole were added to the cultures, instead of adding niridazole directly to the cultures. Such serum and urine were found to be inhibitory for in vitro induction of CTL. The serum and urine had no effect on the effector phase of CTL.
INTRODUCTION In recent studies, niridazole, an anti-helminthic drug used for 12 years in the treatment of schistosomiasis, has been shown to be a potent suppressant of T cell-mediated immune reactions in a number of species. Given orally to mice, niridazole markedly inhibited both granuloma formation around Schistosoma mansoni eggs and the delayed footpad swelling elicited by soluble schistosome egg antigen (Mahmoud & Warren, 1974). Niridazole also delayed the rejection of mouse and rat allografts (Mahmoud, Warren & Webster, 1975; Salaman et al., 1977). When administered to guinea-pigs sensitized to a modified bovine y-globulin, niridazole abolished the delayed cutaneous reactivity and blocked the production of migration inhibition factor (Daniels, Warren & David, 1975). In man, niridazole resulted in reduced or ablating skin reactions to tuberculin, mumps and schistosome antigens (Webster et al., 1975). Antibody responses, however, were only minimally affected by the drug (Pelley et al., 1975). During an investigation of the mechanism of the suppression of the T cell-mediated immune response, it was suggested that niridazole metabolites produced by niridazole administration might be responsible for this activity (Mahmoud et al., 1975; Webster et al., 1975). This hypothesis was supported by the observation that sera and urine, from animals treated with niridazole, blocked the in vitro production of migration inhibition factor, while niridazole itself was inactive (Daniels, Fajardo & David, 1975; Lucas et al., 1977). The studies reported here were undertaken to analyse further the niridazole-induced prolongation of skin allograft survival in mice, as well as to investigate the effect of sera and urine from mice treated with niridazole on the generation of cytotoxic T lymphocytes (CTL) induced in a mixed lymphocyte Correspondence: Dr M. Rollinghoff, Institut fur Medizinische Microbiologie der Universitat Mainz, Hochhaus am Augustusplatz, D-6500 Mainz, West Germany. 0099-9104178/0600-0411$02.00 (©1978 Blackwell Scientific Publications
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W. Solbacli, H. Wagner & M. Rollinghoff
reaction. The data confirm the reversible effect of niridazole in delaying allograft rejection and indicate that the time interval between termination of drug administration and the beginning of graft rejection is about 6 days. Furthermore, the results indicate that sera and urine from niridazole-treated mice are capable of suppressing the in vitro induction of CTL, when given to cultures in their early phase. MATERIALS AND METHODS Mice. CBA/J and BALB/c inbred mice were purchased from GI Bomholtgaard, Ry, Denmark, and used at an age of 8-12 weeks. Niridazole. Niridazole [1-(5-nitro-2-thiazolyl)-2 imidazolodinone], a gift of Ciba-Geigy, Wehr/Baden, West Germany, was suspended in distilled water at a concentration of 5 0 mg/ml. The mice, anaesthetized with ether, received niridazole orally by a plastic tube inserted into the oesophagus, at a dose of about 100 mg/kg. Sera. Blood was obtained from mice by bleeding through the orbit and allowed to stand for 30 min at 4VC. Sera were centrifuged at 3000 g for 20 min. They were not inactivated, and they were diluted 1:10 in culture medium, sterilized through a Millipore filter (pore size 0-22 pm) and stored at - 20'C until used. Sera from mice which had received niridazole daily on at least 7 consecutive days were designated NIR sera. Urine. Urine was reliably obtained from mice by kneading the skin of the neck and at the same time carefully pulling of the tail, thereby triggering a micturition reflex. Urine was processed in a similar way to the serum. Urine from mice which had received niridazole daily on at least 4 consecutive days was designated NIR urine. Skin allografting. Skin grafting of BALB/c tail skin on CBA mice was done according to the method described by Billingham (1961). Tail skin was cut across to give grafts of 1.0 cm2 and these were applied to the dorso-lateral thorax of the recipients and covered with a non-adherent dressing and a bandage. Dressings were removed on day 9, and the grafts were considered rejected when two independent examiners scored more than 80%. of the graft as necrosed. In vitro induction of alloantigen-reactive cytotoxic T lymphocytes. The basic culture system has been described in detail (Rollinghoff et al., 1975; Wagner et al., 1976). In brief: spleen cells were used as source of responding and stimulating cells. Before culture, the stimulating cells received a dose of2000 rad (Philips machine, RT 200) at a dose rate of 320 rad/min. The cells were cultured in multi-dish culture trays (Costar 3524, Costar Cambridge, Massachusetts) using Dulbecco's modified Eagle's medium (DMEM) (Grand Island Biological Co., Grand Island, New York), supplemented with 5 x 10-5M 2-mercaptoethanol and 5% foetal calf serum. CBA responder cells (4 x 106) and BALB/c stimulator cells (106) were cultured in a volume of 2-0 ml in a humidified atmosphere of 10%4 CO2 for 5 days. Then the cytotoxic activity generated was tested in a 51Cr-release assay. To some cultures graded concentrations, of NIR serum or NIR urine were added in a final volume of 0-2 ml culture medium. Cytotoxic assay. The cytotoxic activity of the CTL induced in vitro was tested according to the method described previously (Wagner et al., 1976). In brief: a constant number of ' Cr-labelled target cells (2 x 104) was mixed with graded numbers of attacker CTL in a volume of 1-0 ml DMEM in round bottom plastic tubes (No. 175, Greiner, Nurtingen, West Germany). After 3 hr incubation, the radioactivity of both the supernatant and pellet was determined in a Packard autogamma counter. Percentage specific lysis was calculated according to the formula described elsewhere (Wagner et al., 1976). The standard deviation (s.d.) of triplicate assays was calculated. The data are presented without s.d. since in all experiments listed the s.d. of the percentage lysis was less than 5%. Statistical analysis was performed using the Student's t-test.
RESULTS
Influence of niridazole on murine allograft rejection The effect of niridazole on skin allograft survival was investigated in CBA mice, which had received a BALB/c strain-derived skin graft. The host mice were treated orally with 100 mg/kg niridazole (or with placebo) daily from day -3 to day 9, and were grafted on day 0. As a control, each experimental mouse also received a syngeneic CBA skin graft. The results are detailed in Table 1: the allografts survived for 18-3 + 1 0 days as compared to a mean survival of 10-5 + -14 days in placebo-treated controls. None of the syngeneic control grafts were rejected. The animal mortality was up to two out of a group of nineteen. These data confirm an earlier report by Mahmoud et al. (1975) and clearly demonstrate niridazole to be an immunosuppressive agent in prolonging skin allograft survival. To test whether niridazole may be of use as an immunosuppressant over a longer period of time, CBA mice received doses of 100 mg/kg of niridazole orally for graded periods of time. The drug treatment was started 2 days before transplanting the BALB/c skin grafts and continued until day 9, 12, 15, 18 or 24. Allograft rejection was delayed in all experimental groups (Fig. 1), and a correlation was found to
Immunosuppressive effect of niridazole
413
TABLE 1. Allograft survival in niridazole-treated mice Treatment with niridazole (100 mg/kg)
Origin of skin graft
Host
CBA
CBA CBA BALB/c BALB/c
(days)
Allograft survival time (days+ s.d.)
No. of mice
Mortality
Placebo control -3to9 Placebo control -3 to 9
> 30 >30 10-5+1-4 18-3+ 1-0
19 19 19 19
2/19 0/19 1/19 2/19
exist between the time of termination of niridazole administration (day 9, 12, 15, 18 and 24) and the timepoint at which 50%4 of the grafts were rejected (day 17, 19, 21, 23 and 30). The mean interval was estimated to be 6-20+ 1 50 days. Syngeneic CBA control skin grafts were accepted permanently over an observation period of 40 days. The mortality of the mice of the experimental groups and the controls did not differ; up to two mice per group died during the experiment, mostly due to the repetitive (up to twenty-six times) gastro-oesophagical drug application, which included ether anaesthesia. All surviving mice appeared healthy over the period of observation. These data suggested that the time of allograft rejection can be arbitrarily determined by predetermining the period of niridazole treatment. Effect of niridazole on the capacity of spleen cells to generate cytotoxic T cells in vitro A first set of experiments were designed to find out if spleen cells were altered in their capacity to generate CTL in vitro when the donors of the spleen cells were treated previously with niridazole. CBA mice received niridazole (100 mg/kg) orally on 7 consecutive days. Subsequently spleen cells (4 x 106) from these mice were cultured as responder cells together with 106 BALB/c strain-derived X-irradiated (2000 rad) stimulator cells for 5 days. The cytotoxic activity generated was measured by a 51Cr-release assay, the results of which are given in Table 2. In six independent experiments, niridazole treatment of the mice that had provided the responder cells over 1 to 11 days had not resulted in a significant decrease in the capacity of the spleen cells to generate CTL in vitro. Similarily, no effect was observed when the donor mice received the drug only once during the period from day -7 to -1 (data not shown). Since pre-treatment of donor mice with niridazole had little inhibiting effect on the capacity of spleen cells of such mice to generate alloreactive CTL, it was of interest to test whether niridazole was able to affect significantly the induction of CTL in vitro, when present during the 5 day culture period. Previous studies had suggested that it was not niridazole, which is insoluble in water, but metabolites ofniridazole that were responsible for the reported immunosuppressive activity (Daniels et al., 1975). Therefore, we 10080-
60 40-
1(I)~~~~~~I
020 0
10
12
14
16
18
20 22 24 26 Days after transplantation
28
30
32
34
FIG. 1. Time relationship between survival ofBALB/c skin grafts on CBA hosts and duration of oral niridazole
administration. (o) Group I (n = 40), without niridazole treatment; (o) group II (n = 25), niridazole daily
100 mg/kg from day -2 to 9; (U) group III (n = 6); niridazole from day -2 to 12; (L) group IV (n = 6), days -2 to 15; (A) group V (n = 6), days -2 to 18; and (A) group VI (n = 17), days -2 to 24. C
W. Solbach, H. Wagner C M. Rillinghoff
414
TABLE 2. Effect of niridazole treatment of the donor mice on the capacity of donor mice-derived responder spleen cells to generate cytotoxic T cells in vitro Specific lysis (%)t of Experiment No.:
Pre-treatment of donor mice (CBA) of the responder spleen cells
NaCi (0 9%) on 11 consecutive days Niridazole (100 mg/kg) on 11 consecutive days§
In vitro CTL induction system*
1
2
3
4
5
6
Mean specific lysis (%)
CBA anti-BALB/c
70
68
96
72
97
101
84-0+
CBA anti-BALB/c
46
47
92
59
85
88
69-5+
* 4 x 106 responder spleen cells were cultured together with 106 X-irradiated (2000 rad) BALB/c stimulator cells for 5 days. t Effector to P815 target cell ratio was 30:1. The assay time of the 51Cr-release test was 3 hr. Background lysis of P815 was 11%. t These values did not differ significantly (P> 0 05). § As control, some of the CBA mice received BALB/c skin grafts, similar to group II in Fig. 1, and they all showed an enhanced graft survival.
used serum from mice treated daily for at least 7 consecutive days with doses of 100 mg/kg of niridazole as a source of niridazole metabolites (subsequently abbreviated as NIR serum) and tried to determine the effect of NIR serum on the induction of CTL in vitro. CBA responder cells (4 x 106) were cultured together with 106 X-irradiated (2000 rad) BALB/c stimulator cells in the presence of graded concentrations of NIR serum. As can be seen in Table 3, the presence of NIR-serum during the culture period markedly inhibited the in vitro generation of CTL. Since not only the serum but also the urine of animals treated with niridazole was shown to contain metabolites of the drug (Faigle & Keberle, 1969; Lucas et al., 1977), we also tested the effect of urine obtained from mice which had received the compound (100 mg/kg) daily by mouth for more than 4 days on the induction of CTL in vitro. On the basis of observations on the pharmacodynamics of niridazole (Faigle & Keberle, 1969), we assumed that after 4 days of daily treatment with niridazole a constant concentration of either niridazole metabolites or a niridazole-induced immunosuppressive factor would be reached in the urine. Such urine was designated NIR urine. The results of an experiment in which the effect of graded concentrations of NIR urine on the induction of CTL in vitro was compared with the effect of normal urine are given in Fig. 2. The data showed that NIR-urine inhibited the generation of CTL in vitro in the same way as NIR serum. TABLE 3. Effect of graded concentration of NIR serum on the generation of alloreactive cytotoxic T cells in vitro
Concentration of serum in the culture system (%)*
Specific lysis (%)
NIR serum
Normal mouse serum
50: It
5:1
0 1 2 3
3 2 1 0
99 42 8 6
40 7 2 4
* CBA anti-BALB/c CTL were induced as described in Table 2. t Effector to P815 target cell ratio. Background lysis was
9%.
Immunosuppressive effect of niridazole
415
904
I0
U 21%
Co (n
Volume (%)
FIG. 2. Effect of graded concentrations of NIR urine on the generation of CBA anti-BALB/c cytotoxic T cells in vitro. 4x 106 CBA normal spleen cells were incubated together with 106 X-irradiated BALB/c-derived spleen cells. Culture medium contained graded concentrations of urine, either derived from normal mice (o) or niridazole pre-treated mice (0). Abscissa shows the lysis of P815 target cells. Effector:target cell 50:1. Background lysis was less than 11%. Exact values in parenthesis.
In the next experiment, we tried to determine whether the suppression of CTL induction by NIR serum and NIR urine was due to interference of the early phase (i.e. antigen recognition) or of the late phase (i.e. differentiation of pre-CTL into effective CTL) of the immune reaction by metabolites of niridazole. In order to test for this, NIR serum or NIR urine were added to the culture in vitro at various times after its onset. The final concentration of NIR serum and NIR urine in the cultures was 2% and 1% respectively. As can be seen in Fig. 3, the presence of metabolites of niridazole in the form of NIR serum or NIR urine during the whole culture period of 5 days markedly affected the induction of alloreactive CTL. If, however, the metabolites were present only during the late phase of the culture (day 80-r
60 1.
a
0 0
401-
c
:aTo
I
._
0 ._
20
C)
F i
L---L-
0
I 2
I .I I
I.I I
1. I.
I "-f
M&A
Jg;Z.;W6-
------
4 3 Day of addition of NIR serum or NIR urine to the culture
1
FIG. 3. Effect of adding 2% NIR serum (n) or 1% NIR urine (0) at various times after the onset of a mixed lymphocyte culture on the induction of CBA anti-BALB/c cytotoxicity. The ordinate represents the percentage inhibition of the cytotoxicity generated in a 5 day culture. The percentage inhibition was calculated by comparing the cytotoxicity induced in cultures supplemented with NIR serum (or urine) and normal serum (or urine). The effector:P815 target cell ratio was 50: 1. The cytotoxic activity of control cultures was 63%. Background lysis was 14%.
W. Solbach, H. Wagner & M. Rdi1inghoff
416
TABLE 4. Effect of NIR serum and NIR urine on the cytolytic activity of CTL*
Concentration (%) of: Serum -
Specific lysis (%)t
Urine
Normal serum
-
68
NIR serum
0*5 0 75
65
65
66
77
71
77
1.0
1*5 2-0
30
Normal urine
NIR urine
65
67
62
64
58
57
* In vitro generated CBA anti-BALB/c CTL (106) were incubated at 37'C in 10 ml culture medium containing 1% foetal calf serum and CBA serum or CBA urine in the concentrations indicated. 2 hr later 2x 104 51Cr-labelled P815 target cells were added, the cell mixtures were briefly centrifuged (1 min, 1000 rev/min) and incubated for a further 3 hr.
t Background lysis was 12%.
3-5), they had only a moderate influence on the CTL generation. From these data it is concluded that the metabolites of niridazole appear to interfere with the early phase of the induction of CTL.
Effect of NIR serum and NIR urine on the effector phase of cytotoxic T lymphocytes The cytotoxic activities of a given population of CBA anti-BALB/c CTL was measured against P815 target cells in a 51Cr-release assay for 3 hr under standard conditions. A parallel experiment was set up in the presence of graded concentrations of NIR serum or NIR urine. No effect of NIR serum or NIR urine on the effector phase of CTL was observed, even in concentrations of 3Y% and 2% respectively (Table 4), indicating that metabolites of niridazole did not interfere with the killing mechanism of CTL, not were they toxic for CTL, when present for as long as 5 hr. DISCUSSION In the present study the effect of niridazole on skin allograft survival and the in vitro induction of alloantigen-reactive CTL was tested. Four points appear to be noteworthy. Firstly, enhanced graft survival times were observed after repeated daily doses of 100 mg/kg niridazole, administered by the oral route, thereby confirming earlier studies by Mahmoud et al. (1975) and Salaman et al. (1977). Secondly, kinetic experiments revealed that by selecting the length of time of niridazole treatment, the time of the prolongation of graft survival could be determined. Thus an interval of 5-7 days between the termination of drug treatment and graft rejection was observed, independent of the length of niridazole treatment. Thirdly, the generation of alloreactive CTL in vitro was found to be suppressed, provided serum or urine from mice treated orally with niridazole was present in the culture during the early phase of the immune reaction (day 1-3). On the other hand, both NIR serum and NIR urine had no effect on the cytolytic effector phase of CTL. In contrast to niridazole, other drugs, such as the alkylating agents, steroids, purine and pyrimidine analogues, as well as 6-mercaptopurine and azathioprine, have all failed to enhance the survival of murine skin allografts across the major histocompatibility barrier when administered in non-toxic doses (Santos, 1967, 1972; Schwartz, 1965; Stewart, 1969). Only repeated doses of anti-thymocyte serum and sublethal total body radiation have been described as prolonging graft survival (Mandel & Asofsky, 1968; Medawar, 1963). Therefore, it has been suggested that niridazole may be useful in clinical organ transplantation (Mahmoud & Warren, 1974). Indeed, the low incidence of major side effects in patients with
Immunosuppressive effect ofniridazole
417
schistosomiasis and guinea-worm infestation treated with niridazole (Lucas, Oduntan & Gilles, 1969), as well the ability to programme the prolongation of skin allograft survival by repeated daily oral treatment with the drug (Fig. 1), would seem to recommend niridazole as a potent immunosuppressant in organ transplantation. In keeping with this is the observation that niridazole, when tested in rats either alone or in combination with prednisolone and/or azathioprine, has a marked enhancing effect on the survival ofcardiac allografts. As no toxic effects of the drug have been noted (Salaman et al., 1977), the allograft survival-enhancing effect of niridazole should now be tested in larger animals, such as dogs and primates. Whereas the survival of allogeneic skin grafts was considerably prolonged in niridazole-treated mice, to our surprise spleen cells from such animals reacted almost normally in vitro. In response to allogeneic stimulator cells, spleen cells from such mice were almost invariably able to differentiate into CTL, when compared to control spleen cells (Table 2). A possible explanation for this could be that an in vivo effective niridazole-dependent suppressive activity is washed off during the preparation of the responder lymphocytes for the in vitro culture. A similar dissociation between in vivo and in vitro reactivity was reported by Daniels et al. (1975). These authors had observed that in guinea-pigs niridazole treatment abolished delayed cutaneous reactivity, whereas little or no effect was seen when cells from these animals were tested for migration inhibitory factor (MIF). The mechanism of niridazole-induced suppression of cell-mediated immune reactivity is also of interest. Mahmoud et al. (1975) and Webster et al. (1975) were the first to suggest that it was not niridazole itself but metabolites of niridazole that were responsible for the immunosuppressive effect. They had shown that sera taken from niridazole-treated animals markedly diminished antigen-induced inhibition of sensitized peritoneal exudate cells in vitro, although niridazole itself was inactive. As described before, NIR serum blocked the MIF production but not the MIF action on macrophages. The effect of NIR serum was found to be reversible, in that sensitized lymph node cells, incubated for 24 hr with NIR serum, then washed and re-incubated with antigen in normal serum, produced normal amounts of MIF (Daniels et al., 1975). Recently, Lucas et al. (1977) have reported that the immunosuppressive activity of niridazole can also be detected in the urine of rats and patients receiving niridazole. We now present evidence that NIR serum as well as NIR urine is capable of diminishing the in vitro induction of alloantigen-reactive CTL (Fig. 2 and Table 3). It is interesting to note that the suppression became effective only when NIR serum or NIR urine was present during the first 2-3 days of culture (Fig, 3). Addition to the culture at day 4 had no significant effect on the in vitro induction of alloreactive CTL, nor was there any effect on the capacity of the CTL to lyse appropriate target cells (Table 4). Similarly, Jones et al. (1977) have recently reported that NIR sera and NIR urine dialysates were effective in inhibiting the human mixed lymphocyte reaction, when added before or within 30 min of the initiation of the reaction. From these data, as well as from unpublished data of Vadas, Bernard & Whitelaw (personal communication), who studied the effect of niridazole on delayed-type hypersensitivity, it may be deduced that niridazole acts on cell-mediated immune reactions via its metabolites during the early phase of T-cell sensitization, but not during the late (effector) phase. Whether niridazole metabolites interfere at the level of antigen presentation, or directly at the level of antigen-reactive T cells, remains an open question. What is the nature of the observed immunosuppressive activity? As already stated, niridazole appears to react not by itself, but only by metabolites (Daniels et al., 1975; Lucas et al., 1977). This is in good agreement with earlier data, which have indicated that niridazole, after administration to an animal, is converted rapidly to unknown metabolites, which have a longer half-life than niridazole, presumably because the drug metabolites bind to plasma proteins (Faigle & Keberle, 1969). Daniels et al. (1975) have also shown that the immunosuppressive activity of NIR serum can be removed by dialysis. However, despite the fact that Lucas et al. (1977) succeeded in enriching the suppressive activity 100- to 1000-fold by combined solvent extraction and chromatographic techniques, the substance responsible for the suppressive activity has not, as yet, been chemically identified. This work was supported by Sonderforschungsbereich 107 (Mainz) of the Deutsche Forschungsgemeinschaft. We are grateful to Mrs L. Ptschelinzew and Mr C. Hardt for their expert technical help.
418
W. Solbach, H. Wagner & M. Rillinghoff
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(1977) Inhibition of human mixed lymphocyte reactions by sera and urine dialysates from niridazole-treated rats. Transplantation, 24, 134. LUCAS, S.V., DANIELS, J.C., SCHUBERT, R.D., SIMPSON, J.M., MAHMOUD, A.A.F., WARREN, K.S., DAVID, J.R. & WEBSTER, L.T. (1977) Identification and purification of immunosuppressive activity in the urine or rats and a human patient treated with niridazole. J. Immunol. 118, 418. LUCAS, A.E., ODUNTAN, S.O. & GILLES, H.M. (1969) Niridazole in guinea-worm infection. Ann. N. Y. Acad. Sci. 160, 729. MAHMOUD, A.A.F. & WARREN, K.S. (1974) Anti-inflammatory effects of tartar emetic and niridazole- suppression of schistosoma egg granuloma. J. Immunol. 112, 222. MAHMOUD, A.A.F., WARREN, K.S. & WEBSTER, L.T. (1975) Niridazole. II. A potent long-acting suppressant of cellular hypersensitivity. J. Immunol. 114, 279. MANDEL, M.A. & ASOFSKY, R. (1968) The effects of heterologous anti-thymocyte sera in mice. I. The use of a graft-
to weaken the immunological reaction against skin homografts in mice. Transplantation, 1, 21. PELLEY, R.P., PELLEY, R.J., STAVITSKY, A.B., MAAHMOUD, A.A.F. & WARREN, K.S. (1975) Niridazole, a potent longacting suppressant of cellular hypersensitivity. III. Minimal suppression of antibody responses. .. Immunol. 115, 1477. ROLLINGHOFF, M., PFIZENMAIER, K., TROSTMANN, H. & WAGNER, H. (1975) T-cell proliferation in mixed lymphocyte culture does not necessarily result in the generation of cytotoxic T cells. Europ... Immunol. 5, 560. SALAMAN, J.R., BIRD, M., GODFREY, A.M., JONES, B., MILLAR, D. & MILLER, J. (1977) Prolonged allograft survival with niridazole, azathioprine, and prednisolone. Transplantation, 23, 29. SANTOS, G.W. (1967) Immunosuppressive drugs. Fed. Proc. 26, 906. SANTOS, G.W. (1972) Chemical immunosuppression, Transplantation (eds J.J. Najarian and R.L. Simmons). p. 206. Lea and Febinger, Philadelphia. SCHWARTZ, R.S. (1965) Immunosuppressive drugs. Prog. Allergy, 9, 246. STEWART, P.B. (1969) Failure of 6-mercaptopurine to prolong the survival of skin allografts in mice. Transplantation, 7, 498. WAGNER, H., STARZINSKI-POWITZ, A., PFIZENMAIER, K. & ROLLINGHOFF, M. (1976) Regulation of T-cell-mediated cytotoxic allograft responses. I. Evidence for antigenicspecific suppressor T-cells. Europ. J. Immunol. 6, 873. WEBSTER, L.T., BUTTERWORTH, A.F., MAHMOUD, A.A.F., MNGOLA, E.N. & WARREN, K.S. (1975) Suppression of delayed hypersensitivity in schistosome-infected patients by niridazole. New Engl. . Med. 292, 1144.
