Niridazole-mediated modulation of suppressor cells in Wistar rats

Immunopharmacology, 8 (1984) 61-68

61

Elsevier IMO 00241

Niridazole-Mediated

Modulation of Suppressor Cells in Wistar Rats

K.B. Sainis, M.B. Gadgil and G.P. Phondke RBOH Section, Medical Division, Bhabha Atomic Research Centre, Modular Laboratories. Trombay, Bombay 400 085, India

(Received 13 July 1983; accepted 11 June 1984)

The antischistosomal drug niridazole has been shown to inhibit inductive (Vadas and Bernard, 1981) as well as effector phases of delayed hypersensitivity (Sainis et al., 1983). Furthermore, it also abrogates help for delayed hypersensitivity in antigen-primed animals (Sainis et al., 1983). The effect of this drug on antigen-induced suppression was examined in the present studies. Profound suppression of delayed hypersensitivity to sheep erythrocytes was obtained in Wistar rats given 10s erythrocytes (i.v.) 6 days before the immunizing dose (2 x lo9 erythrocytes, i.p.). When these rats were orally administered niridazole (50 mg/kg) 7 days before the tolerising dose of antigen, suppression of delayed hypersensitivity was not obtained. Splenic lymphocytes of rats given the tolerising dose 6 days earlier adoptively transferred the suppression to inbred recipients. Treatment of these afferent suppressor cells with sera from niridazole-treated unimmunized rats abrogated their function. Likewise, the efferent suppressor cells obtained from fully tolerised rats did not suppress the delayed hypersensitivity when co-transferred with immune lymphocytes, if they were pretreated with niridazole-active serum. The metabolite of niridazole present in this serum seems to impair the suppressor cells functionally. Niridazole may thus prove to be a versatile immunomodulator for effector, helper and suppressor T-cells. Abstract:

Key words:

Niridazole; Suppression of delayed hypersensitivity to sheep erythrocytes; Modulation of suppressor cells

Introduction The anti-schistosomal agent niridazole (NIR) markedly suppresses cell-mediated immunity and this effect is attributed to its dialysable metabolic product(s) present in the urine and sera of treated animals (Mahmoud and Warren, 1974; Daniels et al., 1975a; 1975b; Paterson et al., 1977; Jones et al., 1977; Blumer et al., 1979; Boyd et al., 1981). One such immunoactive metabolite, l-thiocarbamoyl2-imidazolidinone (TCI) has been isolated and it has been shown to affect a variety of cell-mediated immune reactions (Tracy et al., 1980; Tracy et al., 1982; Gautam et al., 1982). Recently we have observed that serum from NIR-treated unimmunized rats inhibited the transfer of delayed hypersensitivity (DH) to sheep erythrocytes (SRBC) by immune spleen cells, suggesting a direct action on effector lymphocytes. NIR may also inhibit the augmenta0162-3109/84/$03.00 0 1984 Elsevier Science Publishers B.V.

tion of antibody response in primed animals (Pelley et al., 1975) or helper function in plaque-forming cell assay (Vadas and Bernard, 1981). The functional impairment of helper T-cells in the DH response following in vitro treatment with serum from niridazole-treated unimmunized rats (NAS) observed by us (Sainis et al., 1983) raised the possibility that NAS might also modulate the activity of other regulatory lymphocytes. Recently Gautam et al. (1982) have observed that TCI, given to B6 mice 1 day before tolerisation, decreased the tolerance to the contact sensitizer dinitrofluorobenzene. The present investigations were, therefore, under-

Abbreviations: DH, delayed hypersensitivity; NIR, niridazole; TCI, I-thiocarbamoyl-2-imidazolidinone; NRS, normal rat serum; NAS, niridazole-active serum; SRBC, sheep erythrocytes; NSL, normal (unimmunized) splenic lymphocytes; ISL, immune splenic lymphocytes; SSL, suppressor splenic lymphocytes.

62 taken to evaluate

the effect of NIR on (u) antigen-

induced suppression of DH and (h) the cells mediating this suppression in Wistar rats.

DH was elicited by injecting front equal

5 days after the immunizing

dose

0.2 ml 50% (v/v) SRBC in one of the

foot-pads. The other foot-pad received an volume of sterile saline. Difference in the

swellings of the two foot-pads 24 h after the eliciting dose was measured by means of a Vernier Calliper

Methods

(least count

0.02 mm).

Animals Effkct of’ NIR on suppression Inbred

Wistar

were used for this study. Antigen SRBC were stored in Alsever’s solution. They were washed repeatedly with sterile saline and used for immunizing rats. Treatment

with niridazole

serum (NAS)

Nai’ve rats were given NIR and were bled 12 days later. Three different preparations of NAS were tested. For each preparation, serum from 15 NIRtreated rats was pooled, heat inactivated and stored at -20°C. This serum has been shown to be very effective in inhibiting the transfer of DH by splenic lymphocytes of immunized rats (Sainis et al., 1983) after treatment of the cells in vitro. Normal serum was collected from untreated control rats of the same sex and age. 5 rats were used for each of the three preparations tested. Suppression

of delayed hypersensitivity

NIR (50 mg/kg) was administered 7 days before the tolerizing dose of SRBC. Immunizing and eliciting doses were given as scheduled. Control groups received either NIR and immunizing and eliciting doses or tolerizing, immunizing and eliciting doses. Transfkr of suppression Two different suppression.

Niridazole (Ambilhar, Ciba Geigy Ltd) was suspended in peanut oil. A dose of 50 mg/kg body weight was administered orally to each rat. Niridazole-active

qf’ DH

rats of either sex, 8- 10 weeks of age

to SRBC

For the suppression of DH to SRBC, lOa SRBC (tolerising dose) were injected i.v. to the rats 6 days before immunization with 2 x log SRBC given intraperitoneally (immunizing dose). The DH response was determined by the foot-pad swelling test (Reuben and Phondke, 1979; Nene et al., 1982).

protocols

and ejkt

of’ NAS

were used for transfer

of

Adoptive transfer Donor rats were given a tolerizing dose of SRBC and 6 days later splenic lymphocytes were obtained from these donors. They were suspended in Eagle’s minimum essential medium and injected i.v. to nai’ve inbred recipients of the same sex (3 x lo7 cells/recipient). The recipients were immunized 2 h after the cell transfer and DH response was elicited 5 days after the cell transfer. Control groups received only immunizing and eliciting doses or splenic lymphocytes of normal (unimmunized) donor rats (NSL) and immunizing and eliciting doses. To assess the effect of NAS on the ability of suppressor splenic lymphocytes (SSL) to transfer the suppression of DH, the SSL were treated with NAS (3 x IO7 cells/ml) at 37°C for 1 h. Treated cells were washed 4 or 5 times, resuspended in Eagle’s medium and transferred i.v. Control group received SSL treated likewise with normal rat serum (NRS). Donor rats were given tolerizing, Passive transfer immunizing and eliciting doses of SRBC. Spleen cell suspension was prepared from those donors showing profound suppression of DH. Another group of donor rats was given immunizing and eli-

63

citing doses. Immunized splenic lymphocytes (ISL) were obtained from these rats. Equal numbers (3 x 107) of SSL and ISL were mixed in 1.0 ml of Eagle’s medium and transferred i.v. to nai’ve recipients. DH was elicited 2 h after cell transfer. Control group was given a mixture of ISL with NSL. To assess the effect of NAS on the cells mediating passive transfer of suppression of DH, SSL were incubated with NAS as described earlier, and after washing 5 times they were mixed with ISL and transferred to recipients. Control group received NRS-treated SSL and ISL. Exhaustive washing of NAS-treated cells

In order to find out whether the active metabolite of niridazole exerted its effect by binding to the surface of the target cells, NAS-treated suppressor cells were washed 2, 5 and 8 times in separate groups and co-transferred with immune lymphocytes. DH was elicited 2 h after the transfer.

ferent days. Their ability to adoptively suppression was assayed.

transfer

Degrees of suppression and statistical analysis

The percentage suppression (% S) of DH was calculated as follows: %

s=

FPSi/ri- F&/m FPSi/,i - FPS,

x 100

where FPSi/,i, FPZ& and FPS, are the foot-pad swellings in the groups of immunized/ISL recipient, suppressed/SSL recipient and unimmunized control rats, respectively. Statistical analysis was carried out by Student’s t test.

Results Eflect of NIR administration on antigen-induced suppression of DH

Dialysis of NAS

NAS obtained 12 days after NIR treatment of rats was dialysed extensively against 30 vol of glass distilled water at 4°C. The dialysate was concentrated by rotary evaporation. The residue was reconstituted in the original volume with sterile Eagle’s medium. Both dialysed NAS and its dialysate were tested for their effect on cells mediating passive transfer of suppression. Kinetics of appearance of immunomodulatory activity in NAS

To study the kinetics of appearance of immunomodulatory activity in NAS, sera were collected from groups of 5 rats each 2,4,6,8, 10 and 12 days after administration of NIR (50 mg/kg). These sera were used for treatment in vitro of immune splenic lymphocytes, which were then transferred iv. to naive recipients of the same sex. In another experiment, afferent suppressor cells were obtained as for adoptive transfer of suppression and these cells were treated in vitro with NAS obtained on dif-

Intravenous injection of a tolerizing dose of SRBC 6 days before the immunizing dose markedly suppressed (78% suppression; p < 0.01) the DH to SRBC in these animals (Fig. 1). When NIR was given 7 days before the tolerizing dose (on day - 13) this antigen-induced suppression of DH was not observed (Fig. 1). NIR under this regimen did not affect the DH response in rats given onIy immunizing and eliciting doses (Fig. 1). The effect of NIR is not attributable to the use of peanut oil as suspending medium (Sainis et al., 1983). NIR could thus inhibit the antigen-induced suppression of DH either by interfering with the induction of suppressor cells or by direct action on the suppressor cells. Effect of NAS on suppressor cells

The antigen-induced suppression of DH to SRBC was transferable to nai’ve recipients, as seen from the data in Figs. 2 and 3. Transfer of splenic lymphocytes from donor rats given immunizing doses on the day of transfer (day 0) resulted in 53% suppression of DH in the recipients (p < 0.05; Fig.

CELLS TRANSFERRED WITH ISL

TREATMENT IN VITRO

_ -m NSL

-B

SSL

-N

SSL

;.

NRS

,’

SSL I

I

05

10

15

FOOT-PAD

Fig.

1. Effect of administration

suppression resentative carried

of DH to SRBC in Wistar experiment

swelling (mean

are shown.

f S.E.M.).

20

25

Cmml

SWELLING

2XlSL

rats. Data

from a rep-

Three such experiments

UNIMMUNIZED

CONTROLS~

were

I 05

4-5 rats. Bars show foot-pad

T.D, 1.D and E.D refer to tolerizing,

and eliciting

--

of NIR on the antigen-induced

out. Each group contained

immunizing

!

I

doses of SRBC, given on the days in-

FOOT-PAD

munizing

did not suppress experiment (Fig.

number

When the SSL were treated in vitro with NAS before transfer, the DH response in the recipients

group.

IN

30

RECIPIENTS(mml

of suppression (day

of DH. -6),

from donors

of SSL/NSL

and

were given eliciting Bars show

foot-pad

were mixed with an equal given immunizing

in vitro as in adoptive ISL was given

doses

2 h later.

swelling

from one out of three experiments

impro-

i.v. to recipients 5 7 recipients

(mean

and

transfer.

f

S.E.M.).

per Data

are shown.

TREATMENT IN VITRO

NSL

was not suppressed (- 3% suppression; Fig. 2). The NIR metabolite in NAS was thus able to inhibit the function of SSL. SSL treated with NRS did not show such an inhibition and were fully suppressive (5 1% suppression; Fig. 2), ruling out the possibility of non-specific effecters in the serum. Similar effect was also observed in passive transfer experiments. When splenic lymphocytes from fully suppressed donors were co-transferred with

--

SSL

-m N RS

SSL

.:, ‘:.:,

y. ,.::‘.;:,;, b

SSL

IMMUNIZED

25

given tolerizing

3 x 10’ SSL/NSL

of ISL, obtained

A mixture which

CECLS

from donors

eliciting doses. SSL were treated

2).

TRANSFERRED

SWELLING

I

r

20

(day 0) and eliciting (day + 5) doses and showing

found suppression.

2). Normal splenic lymphocytes the DH in the adoptive transfer

15

Fig. 3. Effect of NAS on passive transfer SSL were obtained

dicated.

10

CONTROLS

\----I

SUPPRESSED

CONTROLS

w

UNIMMUNIZED

CONTROLS

b r

IO FOOT-PAD

1 2.0

SRBC on day -6.

2 5

SWELLING

Fig. 2. Effect of NAS on the adoptive DH. SSL were obtained

I

I

15

3.0

3.5

IN RECIPIENTS(mm1

transfer

of suppression

of

from donors

given tolerizing

doses of

3 x IO’ SSL/NSL

were transferred

to nai’ve

inbred recipients of the same sex on day 0. Immunizing doses were given to all recipients within 2 h of the adoptive transfer. DH was elicited on day + 5. For treatment

of SSL before trans-

fer, cells were incubated with NAS or NRS at 37°C for I h. They were later washed 4 or 5 times and given i.v. to 5 7 recipients/ group. Bars show foot-pad swelling (mean f from one of the three experiments are shown.

S.E.M.).

Data

ISL, 99% suppression of DH was obtained in the recipients (p < 0.001; Fig. 3). Neither NSL mixed with ISL nor the transfer of a higher dose (6 x 107) of ISL displayed such a suppression in the nai’ve recipients @ > 0.1; Fig. 3). When the SSL used for passive transfer of suppression of DH were pretreated in vitro with NAS, their ability to transfer suppression was abrogated (17% suppression, p > 0.1; Fig. 3). NRS-treated SSL, on the other hand, were fully suppressive (8 1% suppression; Fig. 3). The immunomodulatory

activity

of NAS was ev-

65 TABLE I Modulation of suppressor cells by different preparations of niridazole-active serum Serum for treatment of SSL

NRS NAS NRS NAS NRS NAS

I I II II III III

their activity was still evident. However, after 8 washes the treated cells regained their suppressive ability (Table II). Effect of dialysis on NAS activity

Suppression of DH (%) Adoptive transfer

Passive transfer

55.0 4.2 51.0 -3.0 ND” ND

51.3 2.1 81.0 17.0 93.0 19.8

The immunomodulatory activity of NAS was dialysable. Dialysed NAS was unable to inhibit the transfer of suppression (Table III). The dialysate, on the other hand, inhibited the activity of suppressor cells when the latter were treated with it in vitro in the same manner as undialysed NAS (Table TABLE II

a Not determined. Each preparation of NRS was obtained from 5 rats while that of NAS was obtained from 15 rats. SSL were treated in vitro before transfer as described in the text.

Effect of exhaustive washing on NAS-treated suppressor cells Cells

transferred

ident in all of the three preparations

Treatment of SSL in vitro

No. of washes after treatment

DH response in recipients (mm’)

tested (Table

0 E#ect of exhaustive washing of NAS-treated cells The effect of NAS, however, seemed to depend upon the binding of some active product of niridazole to the suppressor cells. This effect was reversible (Table II). After washing the suppressor cells 2-5 times before transfer, the inhibition of

ISL ISL ISL ISL ISL

+ + + +

SSL SSL SSL SSL

NRS NAS NAS NAS

0.94 2.24 1.47 2.06 2.26 1.36

5 2 5 8

f f f f f f

0.13 0.11 0.14 0.11 0.41 0.13

’ Mean f S.E.M. ISL and SSL were obtained for passive transfer of suppression as described in the text. All washes were in cold tissue culture medium. DH was elicited 2 h after the transfer of cells.

TABLE III Effect of dialysis on the activity of NAS Cells co-transferred with immune cells

Treatment of SSL

DH response in recipients

% Suppression

(mm?

NSL SSL SSL SSL SSL SSL No cell transfer

NRS NAS Dialysed NAS Dialysate of NAS

2.17 1.93 0.97 0.96 1.85 0.79 1.89 1.13

f f f f f f f f

0.07 0.14 0.12 0.13 0.10 0.13 0.14 0.05

115.4 116.3 30.7 132.7 26.9

B Mean f S.E.M. Passive transfer of suppression was carried out using suppressor cells and immune spleen cells. 5 to 7 rats were used per group

66 TABLE

IV

Kinetics

of appearance

NIR-treated transfer

rats.

of immunomodulatory

Inhibition

of transfer

activity

in sera of

of DH and adoptive

of suppression

lective inhibition of the inductive phase of cell-mediated immunity in NIR-treated animals. Serum from NIR-treated animals has been shown to inhibit the passive transfer of DH by immune spleen cells (Sainis et al., 1983). Daniels

Day of collection

Transfer

of NAS for treatment

DH (%)

of SSLjISL

of

Transfer

in vitro

(%)

2

128.8

-64.8

117.1

-40.5

6

105.4

-38.9

8

0.9

-51.1

2.7

-28.2

12 controls

SSL or ISL were treated

with sera from NIR-treated

tained

ISL were used for passive

on different

days.

tigen. However, NAS had no effect on preformed macrophage migration inhibition factor (Daniels et al., 1975b). These data only suggest a possible direct action of NIR-metabohte(s) on the effector T-cells

94.9

100

DH while SSL were used for adoptive

transfer

rats obtransfer

of

of suppression.

5 rats were used per group.

III). Since the dialysate was concentrated at 60°C its active ingredient appears to be heat-stable. Kinetics of appearance ity in NAS

of immunomodulatory

activ-

Table IV shows the inhibition of transfer of DH by ISL following treatment with sera obtained on various days after administration of NIR. Significant inhibition of transfer of DH was obtained following treatment of ISL with sera collected after 8 and 12 days. When suppressor cells were treated with these sera and adoptively transferred, the suppression was not obtained in recipients of suppressor cells treated with NAS obtained days (Table IV).

et al. (1975a) have

observed that NAS inhibited the elaboration of macrophage migration inhibition factor by sensitized lymphocytes when it was added before the an-

suppression

4

NRS-treated

of

after 2, 4, 6, 8 and

12

Discussion Suppression of cell-mediated immunity in niridazole-treated rats is attributed to the immunomodulatory activity of the metabolite(s) of this drug (Daniels et al., 1975a; 1975b; Jones et al., 1977; Blumer et al., 1979; Tracy et al., 1982; Gautam et al., 1982). Vadas and Bernard (1981) have shown se-

which remains to be conclusively established. Vadas and Bernard (198 1) have shown that cells of NIR-treated animals were less efficient in providing helper cell function in the antibody response. In the DH response NAS has also been shown to inhibit the helper cells (Sainis et al., 1983). We have now observed that administration of NIR prevented induction of suppression of DH by subsequent i.v. injection of the antigen. This observation confirms a similar effect of the niridazole metabolite, TCI, on the tolerance to contact sensitization by dinitrofluorobenzene in mice (Gautam et al., 1982). Administration of 10m4 10-i g/kg TCI to B6 mice 1 day before the tolerogen reduced the suppression of contact sensitivity. Our observations, however, further demonstrate that NAS exerts direct inhibitory action on suppressor cells of both afferent and efferent limbs as evidenced from cell transfer experiments (Figs. 2 and 3). Since normal rat serum was ineffective, the action of NAS could be due to a NIR-derived or NIR-induced factor, such as TCI. The effect of NAS on the suppressor cell function probably originates from the binding of its immunoactive metabolite to the target cell surface. Inhibition was observed when NAS-treated suppressor cells were washed 2 to 5 times before transfer. However, exhaustive washing (8 times) resulted in the restoration of their suppressive ability (Table IT). This confirms the finding of Daniels et al. (1975a) on the reversibility of the effect of NAS by exhaustive washing. Earlier reports (Daniels et al., 1975a) showed that serum had to be obtained from guinea pig shortly (14 days) after treatment with NIR to be

67

active. In our experiments, serum collected 12 days after NIR administration has been used. This serum is found to be effective in suppressing the passive transfer of DH by effector lymphocytes as well as in inhibiting the functions of helper cells (Sainis et al., 1983) and suppressor cells. This is an interesting observation in the light of the known concentration-dependent effects of the NIR metabolite, TCI. One possibility is that susceptibility of a given cellmediated immune response varies with antigen and species. Secondly, with the rates of catabolism of NIR being different in difSerent species, the concentrations of TCI in the sera of these animals could vary. Alternatively, the immunomodulatory factor in the serum of NIR-treated rats could be something other than TCI. This factor was dialysable (Table III) and stable at 60°C at which it was concentrated. Furthermore, its effect on suppressor cells was reversed following exhaustive washing (Table II). However, the kinetics of the appearance of the immunomodulatory activity presented a rather intriguing picture. While the suppressor cell (afferent) modulating activity was seen in sera obtained after 2, 4, 6, 8 and 12 days, the inhibition of transfer of DH was seen with sera obtained only after 8 and 12 days (Table IV). All but the last finding discussed above suggest that the effective metabolite could be TCI. Nevertheless, these data suggest that although the threshold concentration of the effective metabolite required to inhibit the transfer of DH and transfer of suppression may be the same, the upper inhibitory limit could be much higher for suppressor cells than for those mediating the transfer of DH. Further experiments are necessary to characterise and identify this factor in our NAS preparations. It is now recognised that cellular interactions leading to the suppression of cell-mediated immunity involve both regulatory T-cells and soluble factors (Rich and Rich, 1974; Claman et al., 1977; Moorhead, 1977; Sy et al., 1980). Considering that NAS is capable of inhibiting the secretion of macrophage migration inhibition factor by Teff cells in DH (Daniels et al., 1975a; 1975b) it seems quite likely that the action of NAS on suppressor cells

may arise from the inhibition of the synthesis or release of suppressor factors. This is a direct demonstration of the modulation of suppressor cells by serum from NIR-treated rats. Taken together with the earlier reports of suppression of cells mediating transfer of DH and helper cells, these observations clearly establish that a niridazole metabolite is a versatile immunomodulator of cell-mediated immunity.

Acknowledgements

We thank Dr. C.V. Bapat, Cancer Research Institute Bombay (now retired) for the generous gift of niridazole, Dr. M. Arunachalam of Deonar Abattoir, Bombay for sheep erythrocytes and Dr. V.R. Shah (Associate Director, Medical Group) and Dr. K. Sundaram (Director, Bio-Medical Group), Bhabha Atomic Research Centre, Bombay for their keen interest and encouragement.

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