- Email: [email protected]
Effect of anticancer drugs on the release of interleukin-3 in vitro
0 1 9 2 - 0 5 6 1 / 9 1 $3.00 + .00 Pergamon Press plc. ,c~1991 International Society for lmmunopharmacology.
Int. J. hnmunopharmac., Vol. 13, No. 8, pp. 1 1 7 7 - 1 1 8 5 . 1991. Printed in Great Britain.
EFFECT OF ANTICANCER DRUGS ON THE RELEASE OF INTERLEUKIN-3 I N VITRO S. I. HASAN* and J. L. TURK Department of Pathology, Royal College of Surgeons of England, 35-43 Lincoln's Inn Fields, London WC2A 3PN, U.K. (Received 20 November 1990 and in final form 26 April 1991)
Abstract - - In this study several anticancer drugs were tested for their effect on the release of interleukin-3
(IL-3) from concanavalin A (Con A) stimulated mouse splenocytes in vitro. When Adriamycin or vincristine were added to the cultures at non-cytotoxic concentrations, the release of IL-3 was inhibited. However, bleomycin, FK156, FK565 and 4-OOH-cyclophosphamide (the in vitro active analogue of the anticancer and immunosuppressive drug cyclophosphamide) did not alter the release of IL-3 under the same conditions. It was confirmed that cyclosporin A inhibited the release of IL-3 in the same experimental system.
lnterleukin-3 or multipotential colony stimulating factor is a glycoprotein produced by activated T-cells (Metcalf, 1987) and supports the growth of pluripotent haematopoietic precursor cells and progenitors of all the haematopoietic lineages to mature granulocytes, macrophages and mast cells (Hapel & Young, 1988). There are some reports that IL-3 may have a role in leukemogenesis under certain conditions (Hapel & Young, 1988; Pebusque et al., 1989) and in animals undergoing graft-vs-host reaction, IL-3 was detected in the serum (Crapper & Schrader, 1986). There is also evidence suggestive of a role for IL-3 in regulation of some inflammatory reactions (Crapper, Vairo, Hamilton, Lewis & Schrader, 1985; Hirasawa, Ohuchi, Kawarasaki, Watanabe & Tsurufiji, 1989) and in the autoimmune disease, systemic lupus erythematosus (Verala, Vidaller, Llorente & Segovia, 1988). It has also been shown that mononuclear cells from sufferers of atopic conditions have increased basal IL-3 mRNA levels (Gauchat, Gauchat, Bettens, De Weck & Stadler, 1990). In spite of the volume of data available the exact role of IL-3 in physiological and pathological states remains unclear. In our investigations on the modulation of cytokine production by anticancer and immunomodulating drugs we have so far reported the effects of these agents on the release of interleukin-1 (IL-1),
interleukin-2 (IL-2), interferon y and tumour necrosis factor a (TNF). The present work investigates the effect of Adriamycin, bleomycin, vincristine, 4-OOH-cyclophosphamide and the anticancer peptides FK565 and FKI56 on the release of IL-3 from Con A stimulated mouse splenocytes in vitro. These drugs are in clinical use to treat haematological malignancies such as leukaemias and lymphomas, as well as other cancers. The release of IL-3 was suppressed by Adriamycin and vincristine at non-cytotoxic concentrations, whilst the other drugs did not exert any effect. The immunosuppressive drug cyclosporin A, which is known to suppress the release of IL-3 (Mantovani, 1982), has been shown to inhibit the release of IL-3 at non-cytotoxic doses in our experimental system.
EXPERIMENTAL PROCEDURES
Animals
Male BALBc mice were purchased from Bantin and Kingman (Hull, U.K.). The mice were fed RM - I(E) rat and mouse expanded diet (Special Diet Services, Witham, Essex) and water a d libitum. The mice were used at 6 - 8 weeks of age.
*Author to whom correspondence should be addressed. 1177
1178
S. I. HASANand J. L. TURK
Drugs and reagents Adriamycin (Farmitalia Carlo Erba) was purchased from commercial sources. Bleomycin was generously provided by Lundbeck Ltd (Luton, U.K.); vincristine was donated by Lederle (Hampshire, U.K.); 4-OOH-cyclophosphamide (this synthetic compound in aqueous solution degrades to 4-OH-cyclophosphamide which is the active metabolite of cyclophosphamide; Colvin, Padgett & Fenselou, 1973) was the gift of Asta-Werke AG (Bielefeld, F.R.G.); FK565 and FKI56 were kindly provided by Fujisawa Pharmaceutical Co. Ltd (Osaka, Japan); cyclosporin A was a gift from Sandoz (Basle, Switzerland). Cyclosporin A was prepared as a stock solution with the use of 0.1 ml ethanol and 0.02 ml Tween 80 made up to 1 mg/ml with RPMI 1640. Further dilutions were made in culture medium. All the other drugs were dissolved in culture medium immediately prior to each experiment. Recombinant murine IL-3 (Genzyme) was purchased commercially; rabbit anti-lL-3 antiserum was the kind gift of Dr Shozo Izui of the Department of Pathology, University of Geneva, Switzerland (Davignon, Kimoto, Kindler, De Kossodo, Vassalli & Izui, 1988). Culture medium For all cell cultures and assays in this work we used RPMI 1640 (Flow Labs, Irvine, U.K.) supplemented with 10% of complement inactivated foetal calf serum (Seralab, Sussex, U.K.) and 50 units/ml penicillin; 50/ag/ml streptomycin (Flow Labs). This formulation is referred to as "culture medium" throughout this work. Cell lines The AC2 cell line used for the IL-3 bioassay was kindly provided by Dr John Garland of the University of Manchester School of Medicine, Manchester, U,K.; the AC2 cells are an IL-3 dependent murine mast cell line (Palacios & Garland, 1984). These cells are grown in culture medium (supplemented with 10°70 of IL-3 rich conditioned medium from WEHI3b cell cultures) at 37°C in humidified atmosphere containing 5% CO2. Production of IL-3 in vitro Con A stimulated mouse splenocyte cultures were used as a source of IL-3. BALBc mice were killed and the spleens removed aseptically. Each spleen was cut up and teased apart over a fine wire gauze, the debris removed and the cells recovered by centrifugation. The erythrocytes were removed by hypo-
osmotic lysis using distilled water. Cell viability was determined by trypan-blue exclusion and the suspension adjusted to 1 × 106 splenocytes/ml of culture medium containing 0.5 tag/ml Con A (Pharmacia, Bucks, U.K.). The cultures were incubated (with and without various non-cytotoxic concentrations of test drug) in plastic, six-well culture plates (Nunc, Denmark) in 2 ml aliquots. After 24 h culture at 37°C the supernatants were collected, centrifuged to remove the ceils and then dialysed against phosphate-buffered saline PBS (Oxoid) 100 ml per 1 ml supernatant at 4°C with one change of PBS at 12 h. The supernatants were then passed through Millipore filters and stored at - 2 0 ° C .
Determination of the toxicity of drugs to Con A stimulated murine splenocytes A wide range of concentrations of the drugs was added to spleen cells (106/ml) together with Con A (0.5 pg/ml) and incubated in plastic six-well culture plates (Nunc) for 24 h. The cells were then collected including the adherent cells, centrifuged and suspended in trypan blue solution (0.02% w/v in PBS). The total number of viable (non-stained) cells were counted and the recovery of viable cells calculated along with the cell recovery figures from drug-free cultures. Three experiments were performed for each drug and the means calculated, along with standard deviations. Concentrations of drugs which resulted in significant reduction in cell viability, in comparison with drug-free cultures, were excluded from the experiments. IL-3 bioassay The IL-3 activity of the supernatants was assayed by their ability to maintain growth of the IL-3 dependent AC2 cell line in a bioassay as described by Palacios & Garland (1984). The AC2 cells were harvested from a growing culture, washed three times with Hank's balanced salt solution to remove IL-3 and suspended in culture medium at 1 x 105 AC2 cells/ml. The test supernatants were titrated in serial doubling dilutions in 96-well flat bottom multiwell plates (Nunc) 100 ~1 volumes using culture medium. A negative control (culture medium alone, i.e. without IL-3) was included in each assay along with a positive control using known amounts of recombinant murine IL-3. Triplicate samples of dilutions were employed throughout. One-hundred microlitres of AC2 cell suspension were then added to each well (1 × l0 t AC2 cells per well) of the culture plate and
Effect of Anticancer Drugs i n c u b a t e d in a h u m i d i f i e d a t m o s p h e r e c o n t a i n i n g 5 % CO: at 37°C for 24 h. In the last 4 h o f culture, each well was pulsed with 1 /~Ci o f [3H] labelled t h y m i d i n e (2 C i / m m o l , A m e r s h a m , U.K.). The cells were then harvested o n t o glass fibre p a p e r ( W h a t m a n ) . T h e radioactivity o n the p a p e r discs was c o u n t e d in a P a c k a r d Tricarb M o d e l 2405 scintillation counter. Results are expressed as m e a n c o u n t s / m i n o f [SH] t h y m i d i n e i n c o r p o r a t i o n of triplicate samples along with s t a n d a r d deviations. The significance o f differences between the m e a n s was evaluated by analysis o f variances a n d by S t u d e n t ' s t-test.
Confirmation o f IL-3 specificity o f the bioassay R a b b i t anti-IL-3 serum ( D a v i g n o n et al., 1988) was used to neutralize the ability o f spleen cell s u p e r n a t a n t s to s u p p o r t the g r o w t h o f A C 2 cells. N o r m a l r a b b i t serum (NRS) was included as a n o n specific serum. Spleen cell s u p e r n a t a n t was preincu b a t e d at 37°C for 1 h with 1 : 20 o f the anti-IL-3 serum, a n d assayed. IL-3 activity was completely abolished as c o m p a r e d with an u n t r e a t e d sample of the same s u p e r n a t a n t , a n d with a f u r t h e r sample which was i n c u b a t e d wth 1 : 20 NRS (Fig. 5).
_3;
1179
25
0
1
2
3
4
5
LOG2 RECIPROCALDILUTION OF SUPERNATANT Fig. 1. Production of IL-3 from mouse spleen cells stimulated with Con A: 5 × I0 ~ splenocytes/ml + 10 ~g/ ml Con A ( O - O ) , 5 × 106 splenocytes/ml + 1 /~g/ml Con A (A - &), 1 × 106 splenocytes/ml + 1 /~g/ml Con A ( O - O ) and 1 × 106 splenocytes/ml + 0.5 gg/ml Con A (Vl-[]) were incubated for 24 h after which the supernatants were collected and assayed for IL-3 activity in the AC2 cell bioassay. Results are mean counts/rain of triplicate samples of dilutions along with standard deviations, rmu IL-3 (O - O) was used from a preparation of 400 u/ml. AC2 cells plus medium alone gave counts/min 739 _+ 199 in this assay.
RESULTS
Production o f IL-3 f r o m mouse spleen cells stimulated with Con A in vitro and the effect o f cyclosporin A M u r i n e spleen cells (5 × 106/ml a n d 1 x 106/ml) were i n c u b a t e d with different c o n c e n t r a t i o n s o f C o n A (10, 1 a n d 0.5 g g / m l ) for 24 h, after which the culture s u p e r n a t a n t s were collected, passed t h r o u g h Millipore filters a n d stored at - 2 0 ° C . T h e IL-3 activity o f these s u p e r n a t a n t s was assayed in the A C 2 cell bioassay along with a s t a n d a r d p r e p a r a t i o n o f r m u IL-3. The assay was p e r f o r m e d using d o u b l i n g dilutions o f the s u p e r n a t a n t s a n d the results are s h o w n in Fig. 1 as c o u n t s / m i n o f [~H] t h y m i d i n e uptake. A dose - response was seen in the p r o d u c t i o n o f IL-3 with different c o n c e n t r a t i o n s of C o n A a n d spleen cells. W h e n cyclosporin A was a d d e d to spleen cell cultures in the presence of optimal c o n c e n t r a t i o n s o f C o n A, n o effect o n IL-3 release was detected. A t s u b o p t i m a l conditions o f IL-3 p r o d u c t i o n (106 s p l e n o c y t e s / m l + 0.5 n g / m l C o n A) cyclosporin A resulted in a d o s e - d e p e n d e n t i n h i b i t i o n o f IL-3 release. Therefore, the same c o n d i t i o n s were a d o p t e d for all experiments to allow the test drugs to exert a detectable effect o n IL-3 release.
12
uj d~-'~ 11 z_x
lO 9
h~
I
I
1
2
|
3
4
LOG2 RECIPROCALDILUTION OF S U P E R N A T A N T
Fig. 2. Effect of cyclosporin A on IL-3 release from spleen cells in vivo. 1 × 106 mouse splenocytes/ml were incubated with Con A (0.5 gg/ml) and 0 (O - O), 1 ng/ml (O - O), 10 ng/ml ( A - A ) , 100 ng/ml ( O - O ) cyclosporin A for 24 h. Supernatants were collected and dialysed to remove the drug and IL-3 activity assayed in AC2 cell bioassay. Results are mean counts/min of triplicate samples of dilutions along with S.D., a(P<0.001), b(P<0.002), c(P<0.05), d(P<0.02). AC2 cells plus medium only gave counts/min 462 _+ 19 and 15 u/ml rmu IL-3 gave 13707 _ 1060 in this assay.
S. |. HASAN and J. L. TURK
1180
c o n c e n t r a t i o n s of 1, 10 a n d 100 n g / m l . A t 10 n g / m l cyclosporin A caused 39°70 inhibition (P<0.05) a n d at 100 n g / m l 7 6 % inhibition (P<0.001) of IL-3 activity o f s u p e r n a t a n t s (see Fig. 2 which is representative o f three experiments).
2O _Zx
15
i!,°
c
b
Effect o f A driamycin on the release o f IL-3 in vitro
UJ~
Various non-cytotoxic concentrations of A d r i a m y c i n were i n c u b a t e d with 1 × l06 m o u s e s p l e n o c y t e s / m l stimulated with C o n A ( 0 . 5 / a g / m l ) for 24 h. A d r i a m y c i n was f o u n d to be non-toxic to mouse splenocytes at c o n c e n t r a t i o n s of 1, 10 and 1 0 0 n g / m l . The s u p e r n a t a n t s were dialysed to remove A d r i a m y c i n before assay for IL-3 activity. At 1, 10 a n d 100 n g / m l A d r i a m y c i n inhibited the release of IL-3 by 38°70 (P<0.02), 49°70 (P<0.02) a n d 30% (P<0.01) respectively as c o m p a r e d with control s u p e r n a t a n t f r o m drug-free culture at half dilution o f s u p e r n a t a n t . The result shows strong inhibition o f IL-3 at low c o n c e n t r a t i o n s o f A d r i a m y c i n in culture a n d the i n h i b i t i o n is not dose dependent. In the assay, due to the m a r k e d decrease in IL-3, the weakly active s u p e r n a t a n t s h a d titrated towards the baseline in the early dilutions. The drug-free control s u p e r n a t a n t h a d diluted relatively slightly at the second dilution (Fig. 3). A series o f three experiments was completed and inhibition of IL-3 release was recorded at 10 and 100 ng c o n c e n t r a t i o n s of A d r i a m y c i n .
>-~
Effect o f vincristine on the release o f IL-3 in vitro
LOG z RECIPROCAL DILUTION
OFSUPERNATANT
Fig. 3. Effect of Adriamycin on the release of IL-3 from mouse splenocytes in vitro. 1 x 10~ mouse splenocyte/ml were incubated with Con A (0.5 ~g/ml) and 0 ( O - O ) , l ng/ml ( O - O ) , 10ng/ml ( A - ~ ) , 100ng/ml ( ~ - ~ ) Adriamycin. Supernatants were harvested after 24 h, dialysed to remove the drug and assayed for IL-3 activity in AC2 cell bioassay. Results are given as mean counts/min of triplicate samples of dilutions along with S.D., a(P<0.001), b(P<0.005), c(P<0.01), d(P<0.002). Medium only gave counts/min 570 _+ 27 and 25 u/ml rmu IL-3 gave 1990 +_ 88 in the assay.
_Zx
a 1 i
~ 2 i
i
3
i
4
d
LOG z RECIPROCAL DILt.MON OF SUPERNATANT Fig. 4. Effect of vincristine on the release of IL-3 in vitro. 1 x 106 mouse spleen cells were incubated with Con A (0.5~g/ml) and 0 ( Q - O ) , 1 ng/ml ( O - © ) , 10ng/ml ( ~ - A ) , 100 ng/ml ( ~ - ~ ) vincristine. Supernatants were harvested after 24 h, dialysed to remove the drug and assayed for IL-3 activity in AC2 cell bioassay. Results shown are mean counts/min of triplicate samples of dilutions with S.D., a(P<0.001), b(P<0.02), c(P<0.05), d(P<0.01). AC2 cells plus medium only gave counts/min 260 + 13 and 25 u/ml rmu IL-3 gave counts/min 7988 + 635 in the assay. T h e i m m u n o s u p p r e s s i v e drug cyclosporin A, which is k n o w n to inhibit IL-3 release u n d e r certain experimental c o n d i t i o n s , was tested at non-cytotoxic
The anticancer alkaloid vincristine was tested over a non-cytotoxic dose range of 1 - 1 0 0 n g / m l (see Table 5). The drug caused a suppression of IL-3 release by 51% (P<0.001) at 10 n g / m l a n d 65% (P<0.001) at 100 n g / m l w h e n c o m p a r e d with the drug-free control at half dilution of s u p e r n a t a n t (see Fig. 2). The experiment was repeated using cells f r o m two more mice a n d similar inhibition of IL-3 was recorded in b o t h experiments.
Effect o f bleomycin on IL-3 release in vitro A n t i c a n c e r antibiotic bleomycin was tested over a wide non-cytotoxic dose range, but did not alter the release o f IL-3 from C o n A stimulated m o u s e splenocytes. T h e experiment was done in triplicate (see Table 1 which is representative o f three separate experiments).
Effect o f 4-OOH-cyclophosphamide on the release o f IL-3 in vitro 4 - O O H - c y c l o p h o s p h a m i d e was tested over a dose range o f 1 - 100 n g / m l , but n o statistically
Effect of Anticancer Drugs
1181
251
2O o?"
-,2 _zx15 i:,-o z~ I,-,,,,lO
JE
5
Medium only
NRS
anti-lL-3
rlL-3
IL-3
IL-3 sup IL-3 sup + NRS + anti-lL-3
60u / ml Fig. 5. Effect of anti-IL-3 serum on the activity of supernatants in the AC2 cell bioassay. The results are mean counts/rain of triplicate samples along with S.D. at half dilution of samples in the assay. AC2 cells in medium gave counts/rain x 10 ~ = 266 ± 36, AC2 cells in 1 : 40 anti-IL-3 serum gave 233 + 76 and AC2 cells in NRS gave a reading of 678 ± 89.
Table 1. Effect of bleomycin on IL-3 release in vitro Bleomycin (ng/ml)
[3HI Thymidine uptake (mean counts/rain _+ S.D.), dilution of supernatant 1/2
0 1 10 100
10210 10920 11603 10323
± ± ± ±
1/4 560 530 744 1093
8257 8303 8740 7677
1/8
± 499 ± 310 _+ 537 ___ 1419
6550 6183 6737 7156
± + ± ±
1/16 1478 334 442 1146
4327 4970 5013 4607
± 345 ± 459 _+ 130 ± 770
1 x 10~ mouse spleen cells/ml were incubated with Con A 0.5 tag/ml and various concentrations of bleomycin for 24 h. Supernatants were dialysed to remove the drug and IL-3 activity assayed in AC2 cell bioassay. The results are given as [rH] thymidine uptake. (Counts/rain) using mean of three samples of dilutions with standard deviations. Negative control of AC2 cells in medium only gave counts/rain 1083 ± 291 and 25 u / m l rmu IL-3 gave counts/min 10533 = 224 in the same assay.
Table 2. Effect of 4-bydroperoxycyclophosphamide on 1L-3 release in vitro
[3H] Thymidine uptake (mean counts/min ± S.D.),
4-hp-cyclophosphamide (ng/ml)
dilution of supernatant 1/2
0 1 10 100
9717 10808 11659 9552
± ± ~ ±
1/4 331 563 934 312
7780 8075 8819 8235
± ± ± ±
1/8 788 600 676 791
5021 5870 5861 5248
± ± ± ±
1/16 662 763 634 357
3294 4509 3909 4132
± ± ± ±
715 798 540 567
1 x l0 n mouse spleen cells/ml incubated with Con A 0.5 t~g/ml and various concentrations of 4-hydroperoxycyclophosphamide for 24 h. Supernatants were dialysed to remove the drug and IL-3 activity assayed in A C 2 cell bioassay. The results are given as mean counts/min of triplicate samples of dilutions with S.D. Negative control of AC2 cells in medium only gave counts/rain 1494 _+ 85 and 25 u / m l rmu IL-3 gave counts/rain 11227 _+ 2943 in the same assay.
S. 1. HASAN and J. L. TURK
1182
Table 3. Effect of FK156 on IL-3 release in vitro FK156 (ng/ml)
[3H] Thymidine uptake (mean c o u n t s / m i n ± S.D.), dilution of supernatant 1/2
0 1 10 100
15389 15361 16169 14375
± ± ± ±
1/4 951 742 722 1619
14541 14766 15587 15675
± ± ± ±
1/8 756 644 254 1460
11212 10713 11318 11287
± ± ± ±
1/16 305 751 204 167
7635 6767 7455 7186
± ± ± ±
970 497 715 166
1 x 10~ murine splenocytes/ml were incubated with Con A 0.5 tag/ml and concentrations of FK156 for 24 h. Supernatants were dialysed to remove the drug and IL-3 activity assayed in AC2 cell bioassay. The results are given as mean counts/rain of triplicate samples of dilutions along with S.D. Negative control of AC2 cells in medium only gave counts/rain 864 ± 133 and 25 u / m l r m u 1L-3 gave c o u n t s / m i n 19181 ± 3297 in the same assay. Table 4. Effect of FK565 on 1L-3 release in vitro FK565 (ng/ml)
[~H] Thymidine uptake (mean c o u n t s / m i n ± S.D.), dilution of superuatant 1/2
0 1 10 100
6749 6499 6021 6395
± ± ± ±
1/4 611 256 412 94
5340 5093 5134 5390
± ± ± ±
1/8 307 204 55 272
4463 3609 3916 3875
± ± ± ±
1/16 704 133 700 79
2739 2819 2997 2837
± ± ± ±
120 500 10 557
1 )< 106 m o u s e splenocytes/ml were incubated with C o n A 0.5 # g / m l and concentrations of FK565 for 24 h. Supernatants were dialysed to remove the drug and IL-3 activity assayed in AC2 cell bioassay. The results are given as mean counts/rain of triplicate samples of dilutions along with S.D. Negative control of AC2 cells in medium only gave c o u n t s / m i n 876 ± 95 and 25 u / m l rmu IL-3 gave c o u n t s / m i n 13210 ± 1050 in the same assay. Table 5. Cytotoxicity of Adriamycin and vincristine on Con A stimulated murine spleen cells
Effect o f FK565 and FKI56 on the release o f lL-3 in
Drug concentration (per ml)
F K 5 6 5 a n d F K I 5 6 a r e low m o l e c u l a r w e i g h t peptides with antimicrobial and anticancer activity. F K 5 6 5 is a s y n t h e t i c a n a l o g u e o f F K I 5 6 . F K I 5 6 is i s o l a t e d f r o m c u l t u r e s o f Streptomyces violaceus. B o t h t h e s e p e p t i d e s h a v e b e e n r e p o r t e d to p o s s e s s immunomodulatory actions (Mine, Watanabe, T a w a r a , Y o k o t a & N i s h i d a , 1983; P a l a c i o s , 1985). FK565 and FKI56 were tested for their effect on C o n A i n d u c e d IL-3 r e l e a s e f r o m m o u s e s p l e n o c y t e s , b u t d i d n o t alter t h e release o f IL-3 (see T a b l e s 3 a n d 4). A series o f t h r e e e x p e r i m e n t s w a s c a r r i e d o u t f o r b o t h d r u g s to c o n f i r m t h e r e s u l t s . T h e a p p a r e n t i n h i b i t i o n o f IL-3 b y A d r i a m y c i n and vincristine could perhaps be the effect of u n d i a l y s e d d r u g in t h e s u p e r n a t a n t i n h i b i t i n g t h e A C 2 cells in t h e IL-3 b i o a s s a y . T o e x c l u d e t h i s possibility the following experiment was performed. One drug-free control supernatant was divided into t h r e e a l i q u o t s . A d r i a m y c i n at 100 n g / m l w a s a d d e d to o n e p o r t i o n a n d v i n c r i s t i n e at t h e s a m e c o n c e n t r a t i o n to t h e s e c o n d , w h i l s t t h e t h i r d p o r t i o n r e m a i n e d u n t r e a t e d . All t h r e e s u p e r n a t a n t s were d i a l y s e d a n d a s s a y e d as n o r m a l a n d s h o w e d t h e s a m e
Zero 1 ng 10 ng 100 ng I #g
% Viable cell recovery _ standard variations Adriamycin
Vincristine
82.5 84.0 81.0 77.6 33.0
82.5 80.0 80.0 82.0 45.3
__. 7 ___6.6 ± 7.3 ± 8 ± 5
___7 ± 7 _ 6.3 ± 10 ± 3.5
1 × l& mouse splenocytes were incubated with 0.5 tag/ml Con A and Adriamycin or vincristine for 24 h. The cells were then collected and viable cells counted using trypan blue, and the percentage of cell recovery calculated. The results are means of three experiments together with standard deviations.
significant effect (P<0.02) was observed on the release of IL-3 from Con A stimulated murine spleen cell c u l t u r e s (see T a b l e 2). A series o f t h r e e e x p e r i m e n t s w a s c o m p l e t e d to c o n f i r m t h e r e s u l t a n d n o e f f e c t w a s n o t e d in a n y e x p e r i m e n t .
vitro
Effect of Anticancer Drugs IL-3 activity on AC2 cells, confirming that Adriamycin and vincristine at 100 ng/ml are effectively removed by the dialysis procedure.
DISCUSSION This study is part of our investigation of the modulation of cytokines by anticancer drugs in which we have previously reported the effect of anticancer drugs on the release of IL-1, IL-2, interferon},, (IFN},) and TNFa (Ahmed, Hamied & Turk, 1987; Ahmed & Turk, 1989; Ahmed, Vander Meide & Turk, 1989; Hamied, Parker & Turk, 1986; Hamied, Parker & Turk, 1987; Hamied & Turk, 1987; Hasan, Ahmed & Turk, 1990). We have now extended the work to include the haematopoietic cytokine IL-3. In the present study, the anticancer antibiotic Adriamycin was found to inhibit the release of IL-3 from Con A stimulated murine splenocytes at noncytotoxic concentrations. It is interesting to observe that Adriamycin enhances the release of IL-I and especially IL-2 (Hamied et al., 1987; Hamied & Turk, 1987; Ehrke, Cohen & Mihich, 1987). There are, however, other reports of this drug inhibiting the release of migration - inhibition-factor (MIF) as reported by Mantovani (1982). Moreover, we have reported that Adriamycin did not affect the release of IFNy (Ahmed et al., 1989). The other anticancer antibiotic, bleomycin, was tested and it failed to exert any effect on the release of IL-3 in our experiments. Although bleomycin is known to enhance the release of IL-1 and IL-2 (Hamied et al., 1986, Hamied & Turk, 1987) it did not affect the release of IFN}, (Ahmed et al., 1989). In clinical usage Adriamycin causes severe myelosuppression, resulting in leukopenia, thrombocytopenia and anaemia, whilst in comparison with other antineoplastic agents, bleomycin causes minimal bone marrow toxicity (Gilman, Goodman & Gilman, 1980). The severe myelosuppression caused by Adriamycin in patients could perhaps be attributed partly to the inhibition of IL-3 by the drug. The differential effects of Adriamycin and bleomycin on haemopoietic functions indicate that these drugs have a highly selective immunomodulating effect. The anticancer drug vincristine, which is an alkaloid derived from the periwinkle plant, resulted in inhibition of the release of IL-3 at non-cytotoxic concentrations. Vincristine has previously been shown to inhibit the release of IL-2 and interferon}, (Ahmed et al., 1987, 1989) which, like IL-3, are products mainly of T-cells. These findings are
1183
corroborated by the observation that vincristine suppresses various T-cell responses (Borel, 1976; Ryoyama, Mace, Ehrke & Mihich, 1982). Cyclophosphamide, which is used for the treatment of leukaemias, lymphomas, multiple myeloma, as well as other turnouts, and in graft-vshost disease has been shown to exert a wide range of immunomodulatory effects. The in vitro active analogue 4-OOH-cyclophosphamide was tested, but it did not alter the release of IL-3 in splenocyte cultures. Previous work has shown that 4-OOHcyclophosphamide at non-cytotoxic levels inhibits the release of IL-2 (Hamied et al., 1987) but does not affect the release of IFN}' (Ahmed et al., 1989) from splenocytes in vitro. Moreover, 4-OOHcyclophosphamide did not alter the release of IL-1 and TNF from macrophages (Hamied et al., 1987; Hasan et al., 1990). In another work, Kaufman, Metelemann, Diamanstein & Hahn (1984) have shown that 4-OOH-cyclophosphamide did not affect the release of IL-1 or IL-2 in vitro from Listeria monocytogenes immune T-cells up to a concentration of 3 tag/ml. It is notable that amongst all the cytokines tested in our studies (IL-I, IL-2, TNFcr, IFNy and IL-3) only IL-2 was modulated by 4-OOH-cyclophosphamide and its release suppressed. It is likely, therefore, that the immunosuppressive effects of cyclophosphamide are mediated mainly by mechanisms other than cytokine modulation. In the present study, we tested the experimental anticancer and immunomodulatory peptides FK565 and FK156 for their effect on IL-3 release. These peptides did not alter the release of IL-3 from spleen cell cultures. In previous work we have reported enhancement of IL-1 by FK565 and FKI56 (Ahmed & Turk, 1989). In that same work, suppression of IL2 release has been reported from spleen cell cultures in vitro. FK565 has been shown to cause macrophage activation in vivo (Watanabe, Tawara, Mine & Kikuchi, 1985) and to enhance the release of TNFa from macrophages in vitro (Hasan et al., 1990). In a further study, these peptides did not alter the release of IFNy (Ahmed et al., 1989). These findings would seem to point to the possibility that these immunoactive anticancer peptides are selective in their action on the various cytokines. Cyclosporin A, an immunosuppressive agent which has been reported to inhibit the release of IL-3 splenocytes (Palacios, 1985), was included in this investigation to confirm the results of these experiments. Cyclosporin A, when added to murine spleen cell cultures stimulated with suboptimal concentrations of Con A, decreased the release of
1184
S. 1. HASAN and J. L. TURK
IL-3 in the supernatant. It is interesting to note that cyclosporin A has an established place in the prevention and treatment o f graft-vs-host disease which is a condition k n o w n to be associated with elevation o f serum IL-3 levels in experimental animals ( C r a p p e r & Schrader, 1986). The present findings may contribute to a better u n d e r s t a n d i n g o f the m o d e o f action o f anticancer drugs, and o f the pathophysiological role o f IL-3. The finding o f suppression o f IL-3 by A d r i a m y c i n
and vincristine is i m p o r t a n t , as IL-3 exerts some control on haemopoiesis and immunity. These drugs, in therapeutic doses, exert toxic effects on these functions. Acknowledgements - - The funding for this work was provided by the Ministry of Science and Technology of the Government of Pakistan. The authors are grateful to Dr John Garland for the gift of cell lines and helpful advice. We also thank Mrs June Saxby for typing the manuscript.
REFERENCES
AHMED, K., HAMIED, T. A. A. & TURK, J. L. (1987). Inhibition of interleukin-2 by vincristine and vinblastine. lmmunopharmac, lmmunotoxic., 9, 391 - 407. AHMED, K. & TURK, J. L. (1989). Effect of anticancer agents neothramycin, aclacinomycin, FK565 and FK156 on the release of interleukin-2 and interleukin-1 in vitro. Cancer Immun. Immunother., 28, 8 7 - 92. AHMED, K., VAN DER ME1DE, P. H. & TURK, J. L. (1989). Effect of anticancer drugs on the release of interferon), in vitro. Cancer Immun. Immunother., 30, 213-218. BOREL, J. F. (1976). Comparative study of in vitro and in vivo drug effects on cell-mediated cytotoxicity. Immunology, 31, 631-641. COLV1N, M., PADGETT, C. A. & FENSELOU,C. (1973). A biologically active metabolite of cyclophosphamide. Cancer Res., 33, 915-918. CRAPPER, R. M. & SCHRADER,J. W. (1986). Evidence for the in vivo production and release into the serum of a T cell lymphokine, persisting-cell stimulating factor (PSF), during graft-versus-host reactions. Immunology, 57, 553 - 558. CRAPPER, R. M., VA1RO, G., HAMILTON~ J. A.~ LEWIS, I. C. & SCHRADER, J. W. (1985). Stimulation of bone marrowderived and peritoneal macrophages by a T lymphocyte-derived hemopoietic growth factor, persisting cell-stimulating factor. Blood, 66, 859-866. DAVIGNON, J. L., KIMOTO, M., KINDLER, V., DE KOSSODO, S., VASSALLI,P. & IZU1, S. (1988). Selective production of interleukin 3 (IL-3) and granulocyte - macrophage colony-stimulating factor (GM-CSF) in vitro by murine L3T4 ~ T cells: lack of spontaneous IL-3 and GM-CSF production by Ly-2-/L3T4-Ipr subset. Eur. J. Immun., 18, 1367- 1372. EHRKE, M. J., COHEN, S. A. & MIHIC'H, E. (1982). Selective effects of Adriamycin on murine host defence systems. Immunology, 65, 5 5 - 77. GAUCHAT, D., GAUCHAT, J. F., BETTENS, F., DE WECK, A. L. & STADLER, B. M. (1990). Cytokine gene expression in atopics: effect of IL-4 on IL-I/3 and IL-6 mRNA levels. Eur. Cytokine Nt., 1, 85-90. GILMAN, A. G., GOODMAN, L. S. & GILMAN, A. (1980). The Pharmacological Basis o f Therapeutics, 6th Edn, pp. 1293 - 1295. Macmillan, New York. HAMIED, T. A. A., PARKER, D. & TURK, J. L. (1986). Potentiation of release of interleukin 2 by bleomycin. Immunopharmacology, 12, 127- 134. HAMIED, T. A. A., PARKER,D. & TURK, J. L. (1987). Effect of Adriamycin, 4-hydroperoxycyclophosphamideand ASTAZ 7557 (Inn Mafosfamide) on the release of IL-I and IL-2 in vitro. Int. J. Immunopharmac., 9, 355-361. HAMIED, T. A. A. & TURK, J. L. (1987). Enhancement of interleukin 2 release in rats by treatment with bleomycin and Adriamycin in vivo. Cancer Immun. lmmunother., 25, 245-249, HAPEL, A. J. & YOUNG, I. G. (1987). Abnormal expression of interleukin 3 and leukaemia. Anticancer Res., 7, 661 -668. HAPEL, A. J. & YOUNG, I. Q. (1988). Molecular biology of interleukin-3. Lymphokines, (ed. Schrader, J. W.) Vol. 15, pp. 9 1 - 26. Academic Press, New York. HASAN, S. I., AHMED, K. & TURK, J. L. (1990). Effect of anticancer drugs on the release of tumour necrosis factors in vitro. Cancer Immun. Immunother., 30, 363 - 366. HIRASAWA, N., OHUCH1, K., KAWARASAKI,K., WATANABE, M. & TSURUFIJI, S. (1989). Occurrence of histamineproduction-increasing factor in postanaphylactic phase of allergic inflammation. Int. Arch. Allergy appl. Immun., 88, 386- 393.
Effect of Anticancer Drugs
1185
KAUFMAN, S. H. E., METELEMANN, C., DIAMANSTEIN, T. & HAHN, H. (1984). Resistance to 4hydroperoxycyclophosphamideof T cells involved in cell mediated antibacterial immunity. Int. J. lmmunopharmac., 6, 8 1 - 8 5 . MANTOVANI,A. (1982). Advances in Pharmacology and Chemotherapy, the Interaction o f Cancer Chemotherapy Agents with Mononuclear Phagocytes, Vol. 19, pp. 35-66. Academic Press, New York. METCALF, D. (1987). The molecular control of normal and leukaemic granulocytes and macrophages. Proc. R. Soc. Lond., B230, 389- 423. MINE, Y. WATANABE, Y., TAWARA, S., YOKOTA, Y. & N1SHIDA, M. (1983), lmmunoactive peptides FKI56 and FK565; enhancement of host defence mechanisms against infection. ]. Antibiot. (Tokyo), 36, 1059- 1066. PALA(IOS, R. (1985). Cyclosporin A inhibits antigen and leclin-induced but not constitutive production of interleukin 3. Eur. J. Immun., 15, 204-206. PALACIOS, R. & GARLAND, J. (1984). Distinct mechanisms may account for the growth-promoting activity of interleukin 3 on cells of lymphoid and myeloid origin. Proc. natn. Acad. Sci. U.S.A., 81, 1208- 1211. PEBtJS,:.)UE, M. J., FAY, C., LAVAC;E, M., SEMPERE, C., SAELAND, S., CAtJX, C. & MANNONI, P. (1989). Recombinant human IL-3 and G-CSF act synergistically in stimulating the growth acute myeloid leukemia cells. Leukemia, 3, 200 - 205. RVOVAMA, K., MACE, K., EHRKE, M. J. & MrHICH, E. (1982). The differential sensitivities of T cell immune functions to vincristine and vinblastine. Int. J. Immunopharmac., 4, 187- 193. VERALA, A., VIDALLER,A., LLORENTE, L. & SEGOVlA, J. D. A. (1988). Presence of an IL-3-producing suppressor T cell resistant to cyclosporin A in the peripheral blood of patients with systemic lupus erythematosus. Clin. exp. lmmun., 73, 424 - 429. WATANABI2, Y., TAWARA,S., M~NE, Y. & KIKUCHI, H. (1985). Immunoactive peptides FKI56 and FK565 - - activation of mouse macrophages. J. Antibiot. (Tokyo), 38, 1781.
Int. J. hnmunopharmac., Vol. 13, No. 8, pp. 1 1 7 7 - 1 1 8 5 . 1991. Printed in Great Britain.
EFFECT OF ANTICANCER DRUGS ON THE RELEASE OF INTERLEUKIN-3 I N VITRO S. I. HASAN* and J. L. TURK Department of Pathology, Royal College of Surgeons of England, 35-43 Lincoln's Inn Fields, London WC2A 3PN, U.K. (Received 20 November 1990 and in final form 26 April 1991)
Abstract - - In this study several anticancer drugs were tested for their effect on the release of interleukin-3
(IL-3) from concanavalin A (Con A) stimulated mouse splenocytes in vitro. When Adriamycin or vincristine were added to the cultures at non-cytotoxic concentrations, the release of IL-3 was inhibited. However, bleomycin, FK156, FK565 and 4-OOH-cyclophosphamide (the in vitro active analogue of the anticancer and immunosuppressive drug cyclophosphamide) did not alter the release of IL-3 under the same conditions. It was confirmed that cyclosporin A inhibited the release of IL-3 in the same experimental system.
lnterleukin-3 or multipotential colony stimulating factor is a glycoprotein produced by activated T-cells (Metcalf, 1987) and supports the growth of pluripotent haematopoietic precursor cells and progenitors of all the haematopoietic lineages to mature granulocytes, macrophages and mast cells (Hapel & Young, 1988). There are some reports that IL-3 may have a role in leukemogenesis under certain conditions (Hapel & Young, 1988; Pebusque et al., 1989) and in animals undergoing graft-vs-host reaction, IL-3 was detected in the serum (Crapper & Schrader, 1986). There is also evidence suggestive of a role for IL-3 in regulation of some inflammatory reactions (Crapper, Vairo, Hamilton, Lewis & Schrader, 1985; Hirasawa, Ohuchi, Kawarasaki, Watanabe & Tsurufiji, 1989) and in the autoimmune disease, systemic lupus erythematosus (Verala, Vidaller, Llorente & Segovia, 1988). It has also been shown that mononuclear cells from sufferers of atopic conditions have increased basal IL-3 mRNA levels (Gauchat, Gauchat, Bettens, De Weck & Stadler, 1990). In spite of the volume of data available the exact role of IL-3 in physiological and pathological states remains unclear. In our investigations on the modulation of cytokine production by anticancer and immunomodulating drugs we have so far reported the effects of these agents on the release of interleukin-1 (IL-1),
interleukin-2 (IL-2), interferon y and tumour necrosis factor a (TNF). The present work investigates the effect of Adriamycin, bleomycin, vincristine, 4-OOH-cyclophosphamide and the anticancer peptides FK565 and FKI56 on the release of IL-3 from Con A stimulated mouse splenocytes in vitro. These drugs are in clinical use to treat haematological malignancies such as leukaemias and lymphomas, as well as other cancers. The release of IL-3 was suppressed by Adriamycin and vincristine at non-cytotoxic concentrations, whilst the other drugs did not exert any effect. The immunosuppressive drug cyclosporin A, which is known to suppress the release of IL-3 (Mantovani, 1982), has been shown to inhibit the release of IL-3 at non-cytotoxic doses in our experimental system.
EXPERIMENTAL PROCEDURES
Animals
Male BALBc mice were purchased from Bantin and Kingman (Hull, U.K.). The mice were fed RM - I(E) rat and mouse expanded diet (Special Diet Services, Witham, Essex) and water a d libitum. The mice were used at 6 - 8 weeks of age.
*Author to whom correspondence should be addressed. 1177
1178
S. I. HASANand J. L. TURK
Drugs and reagents Adriamycin (Farmitalia Carlo Erba) was purchased from commercial sources. Bleomycin was generously provided by Lundbeck Ltd (Luton, U.K.); vincristine was donated by Lederle (Hampshire, U.K.); 4-OOH-cyclophosphamide (this synthetic compound in aqueous solution degrades to 4-OH-cyclophosphamide which is the active metabolite of cyclophosphamide; Colvin, Padgett & Fenselou, 1973) was the gift of Asta-Werke AG (Bielefeld, F.R.G.); FK565 and FKI56 were kindly provided by Fujisawa Pharmaceutical Co. Ltd (Osaka, Japan); cyclosporin A was a gift from Sandoz (Basle, Switzerland). Cyclosporin A was prepared as a stock solution with the use of 0.1 ml ethanol and 0.02 ml Tween 80 made up to 1 mg/ml with RPMI 1640. Further dilutions were made in culture medium. All the other drugs were dissolved in culture medium immediately prior to each experiment. Recombinant murine IL-3 (Genzyme) was purchased commercially; rabbit anti-lL-3 antiserum was the kind gift of Dr Shozo Izui of the Department of Pathology, University of Geneva, Switzerland (Davignon, Kimoto, Kindler, De Kossodo, Vassalli & Izui, 1988). Culture medium For all cell cultures and assays in this work we used RPMI 1640 (Flow Labs, Irvine, U.K.) supplemented with 10% of complement inactivated foetal calf serum (Seralab, Sussex, U.K.) and 50 units/ml penicillin; 50/ag/ml streptomycin (Flow Labs). This formulation is referred to as "culture medium" throughout this work. Cell lines The AC2 cell line used for the IL-3 bioassay was kindly provided by Dr John Garland of the University of Manchester School of Medicine, Manchester, U,K.; the AC2 cells are an IL-3 dependent murine mast cell line (Palacios & Garland, 1984). These cells are grown in culture medium (supplemented with 10°70 of IL-3 rich conditioned medium from WEHI3b cell cultures) at 37°C in humidified atmosphere containing 5% CO2. Production of IL-3 in vitro Con A stimulated mouse splenocyte cultures were used as a source of IL-3. BALBc mice were killed and the spleens removed aseptically. Each spleen was cut up and teased apart over a fine wire gauze, the debris removed and the cells recovered by centrifugation. The erythrocytes were removed by hypo-
osmotic lysis using distilled water. Cell viability was determined by trypan-blue exclusion and the suspension adjusted to 1 × 106 splenocytes/ml of culture medium containing 0.5 tag/ml Con A (Pharmacia, Bucks, U.K.). The cultures were incubated (with and without various non-cytotoxic concentrations of test drug) in plastic, six-well culture plates (Nunc, Denmark) in 2 ml aliquots. After 24 h culture at 37°C the supernatants were collected, centrifuged to remove the ceils and then dialysed against phosphate-buffered saline PBS (Oxoid) 100 ml per 1 ml supernatant at 4°C with one change of PBS at 12 h. The supernatants were then passed through Millipore filters and stored at - 2 0 ° C .
Determination of the toxicity of drugs to Con A stimulated murine splenocytes A wide range of concentrations of the drugs was added to spleen cells (106/ml) together with Con A (0.5 pg/ml) and incubated in plastic six-well culture plates (Nunc) for 24 h. The cells were then collected including the adherent cells, centrifuged and suspended in trypan blue solution (0.02% w/v in PBS). The total number of viable (non-stained) cells were counted and the recovery of viable cells calculated along with the cell recovery figures from drug-free cultures. Three experiments were performed for each drug and the means calculated, along with standard deviations. Concentrations of drugs which resulted in significant reduction in cell viability, in comparison with drug-free cultures, were excluded from the experiments. IL-3 bioassay The IL-3 activity of the supernatants was assayed by their ability to maintain growth of the IL-3 dependent AC2 cell line in a bioassay as described by Palacios & Garland (1984). The AC2 cells were harvested from a growing culture, washed three times with Hank's balanced salt solution to remove IL-3 and suspended in culture medium at 1 x 105 AC2 cells/ml. The test supernatants were titrated in serial doubling dilutions in 96-well flat bottom multiwell plates (Nunc) 100 ~1 volumes using culture medium. A negative control (culture medium alone, i.e. without IL-3) was included in each assay along with a positive control using known amounts of recombinant murine IL-3. Triplicate samples of dilutions were employed throughout. One-hundred microlitres of AC2 cell suspension were then added to each well (1 × l0 t AC2 cells per well) of the culture plate and
Effect of Anticancer Drugs i n c u b a t e d in a h u m i d i f i e d a t m o s p h e r e c o n t a i n i n g 5 % CO: at 37°C for 24 h. In the last 4 h o f culture, each well was pulsed with 1 /~Ci o f [3H] labelled t h y m i d i n e (2 C i / m m o l , A m e r s h a m , U.K.). The cells were then harvested o n t o glass fibre p a p e r ( W h a t m a n ) . T h e radioactivity o n the p a p e r discs was c o u n t e d in a P a c k a r d Tricarb M o d e l 2405 scintillation counter. Results are expressed as m e a n c o u n t s / m i n o f [SH] t h y m i d i n e i n c o r p o r a t i o n of triplicate samples along with s t a n d a r d deviations. The significance o f differences between the m e a n s was evaluated by analysis o f variances a n d by S t u d e n t ' s t-test.
Confirmation o f IL-3 specificity o f the bioassay R a b b i t anti-IL-3 serum ( D a v i g n o n et al., 1988) was used to neutralize the ability o f spleen cell s u p e r n a t a n t s to s u p p o r t the g r o w t h o f A C 2 cells. N o r m a l r a b b i t serum (NRS) was included as a n o n specific serum. Spleen cell s u p e r n a t a n t was preincu b a t e d at 37°C for 1 h with 1 : 20 o f the anti-IL-3 serum, a n d assayed. IL-3 activity was completely abolished as c o m p a r e d with an u n t r e a t e d sample of the same s u p e r n a t a n t , a n d with a f u r t h e r sample which was i n c u b a t e d wth 1 : 20 NRS (Fig. 5).
_3;
1179
25
0
1
2
3
4
5
LOG2 RECIPROCALDILUTION OF SUPERNATANT Fig. 1. Production of IL-3 from mouse spleen cells stimulated with Con A: 5 × I0 ~ splenocytes/ml + 10 ~g/ ml Con A ( O - O ) , 5 × 106 splenocytes/ml + 1 /~g/ml Con A (A - &), 1 × 106 splenocytes/ml + 1 /~g/ml Con A ( O - O ) and 1 × 106 splenocytes/ml + 0.5 gg/ml Con A (Vl-[]) were incubated for 24 h after which the supernatants were collected and assayed for IL-3 activity in the AC2 cell bioassay. Results are mean counts/rain of triplicate samples of dilutions along with standard deviations, rmu IL-3 (O - O) was used from a preparation of 400 u/ml. AC2 cells plus medium alone gave counts/min 739 _+ 199 in this assay.
RESULTS
Production o f IL-3 f r o m mouse spleen cells stimulated with Con A in vitro and the effect o f cyclosporin A M u r i n e spleen cells (5 × 106/ml a n d 1 x 106/ml) were i n c u b a t e d with different c o n c e n t r a t i o n s o f C o n A (10, 1 a n d 0.5 g g / m l ) for 24 h, after which the culture s u p e r n a t a n t s were collected, passed t h r o u g h Millipore filters a n d stored at - 2 0 ° C . T h e IL-3 activity o f these s u p e r n a t a n t s was assayed in the A C 2 cell bioassay along with a s t a n d a r d p r e p a r a t i o n o f r m u IL-3. The assay was p e r f o r m e d using d o u b l i n g dilutions o f the s u p e r n a t a n t s a n d the results are s h o w n in Fig. 1 as c o u n t s / m i n o f [~H] t h y m i d i n e uptake. A dose - response was seen in the p r o d u c t i o n o f IL-3 with different c o n c e n t r a t i o n s of C o n A a n d spleen cells. W h e n cyclosporin A was a d d e d to spleen cell cultures in the presence of optimal c o n c e n t r a t i o n s o f C o n A, n o effect o n IL-3 release was detected. A t s u b o p t i m a l conditions o f IL-3 p r o d u c t i o n (106 s p l e n o c y t e s / m l + 0.5 n g / m l C o n A) cyclosporin A resulted in a d o s e - d e p e n d e n t i n h i b i t i o n o f IL-3 release. Therefore, the same c o n d i t i o n s were a d o p t e d for all experiments to allow the test drugs to exert a detectable effect o n IL-3 release.
12
uj d~-'~ 11 z_x
lO 9
h~
I
I
1
2
|
3
4
LOG2 RECIPROCALDILUTION OF S U P E R N A T A N T
Fig. 2. Effect of cyclosporin A on IL-3 release from spleen cells in vivo. 1 × 106 mouse splenocytes/ml were incubated with Con A (0.5 gg/ml) and 0 (O - O), 1 ng/ml (O - O), 10 ng/ml ( A - A ) , 100 ng/ml ( O - O ) cyclosporin A for 24 h. Supernatants were collected and dialysed to remove the drug and IL-3 activity assayed in AC2 cell bioassay. Results are mean counts/min of triplicate samples of dilutions along with S.D., a(P<0.001), b(P<0.002), c(P<0.05), d(P<0.02). AC2 cells plus medium only gave counts/min 462 _+ 19 and 15 u/ml rmu IL-3 gave 13707 _ 1060 in this assay.
S. |. HASAN and J. L. TURK
1180
c o n c e n t r a t i o n s of 1, 10 a n d 100 n g / m l . A t 10 n g / m l cyclosporin A caused 39°70 inhibition (P<0.05) a n d at 100 n g / m l 7 6 % inhibition (P<0.001) of IL-3 activity o f s u p e r n a t a n t s (see Fig. 2 which is representative o f three experiments).
2O _Zx
15
i!,°
c
b
Effect o f A driamycin on the release o f IL-3 in vitro
UJ~
Various non-cytotoxic concentrations of A d r i a m y c i n were i n c u b a t e d with 1 × l06 m o u s e s p l e n o c y t e s / m l stimulated with C o n A ( 0 . 5 / a g / m l ) for 24 h. A d r i a m y c i n was f o u n d to be non-toxic to mouse splenocytes at c o n c e n t r a t i o n s of 1, 10 and 1 0 0 n g / m l . The s u p e r n a t a n t s were dialysed to remove A d r i a m y c i n before assay for IL-3 activity. At 1, 10 a n d 100 n g / m l A d r i a m y c i n inhibited the release of IL-3 by 38°70 (P<0.02), 49°70 (P<0.02) a n d 30% (P<0.01) respectively as c o m p a r e d with control s u p e r n a t a n t f r o m drug-free culture at half dilution o f s u p e r n a t a n t . The result shows strong inhibition o f IL-3 at low c o n c e n t r a t i o n s o f A d r i a m y c i n in culture a n d the i n h i b i t i o n is not dose dependent. In the assay, due to the m a r k e d decrease in IL-3, the weakly active s u p e r n a t a n t s h a d titrated towards the baseline in the early dilutions. The drug-free control s u p e r n a t a n t h a d diluted relatively slightly at the second dilution (Fig. 3). A series o f three experiments was completed and inhibition of IL-3 release was recorded at 10 and 100 ng c o n c e n t r a t i o n s of A d r i a m y c i n .
>-~
Effect o f vincristine on the release o f IL-3 in vitro
LOG z RECIPROCAL DILUTION
OFSUPERNATANT
Fig. 3. Effect of Adriamycin on the release of IL-3 from mouse splenocytes in vitro. 1 x 10~ mouse splenocyte/ml were incubated with Con A (0.5 ~g/ml) and 0 ( O - O ) , l ng/ml ( O - O ) , 10ng/ml ( A - ~ ) , 100ng/ml ( ~ - ~ ) Adriamycin. Supernatants were harvested after 24 h, dialysed to remove the drug and assayed for IL-3 activity in AC2 cell bioassay. Results are given as mean counts/min of triplicate samples of dilutions along with S.D., a(P<0.001), b(P<0.005), c(P<0.01), d(P<0.002). Medium only gave counts/min 570 _+ 27 and 25 u/ml rmu IL-3 gave 1990 +_ 88 in the assay.
_Zx
a 1 i
~ 2 i
i
3
i
4
d
LOG z RECIPROCAL DILt.MON OF SUPERNATANT Fig. 4. Effect of vincristine on the release of IL-3 in vitro. 1 x 106 mouse spleen cells were incubated with Con A (0.5~g/ml) and 0 ( Q - O ) , 1 ng/ml ( O - © ) , 10ng/ml ( ~ - A ) , 100 ng/ml ( ~ - ~ ) vincristine. Supernatants were harvested after 24 h, dialysed to remove the drug and assayed for IL-3 activity in AC2 cell bioassay. Results shown are mean counts/min of triplicate samples of dilutions with S.D., a(P<0.001), b(P<0.02), c(P<0.05), d(P<0.01). AC2 cells plus medium only gave counts/min 260 + 13 and 25 u/ml rmu IL-3 gave counts/min 7988 + 635 in the assay. T h e i m m u n o s u p p r e s s i v e drug cyclosporin A, which is k n o w n to inhibit IL-3 release u n d e r certain experimental c o n d i t i o n s , was tested at non-cytotoxic
The anticancer alkaloid vincristine was tested over a non-cytotoxic dose range of 1 - 1 0 0 n g / m l (see Table 5). The drug caused a suppression of IL-3 release by 51% (P<0.001) at 10 n g / m l a n d 65% (P<0.001) at 100 n g / m l w h e n c o m p a r e d with the drug-free control at half dilution of s u p e r n a t a n t (see Fig. 2). The experiment was repeated using cells f r o m two more mice a n d similar inhibition of IL-3 was recorded in b o t h experiments.
Effect o f bleomycin on IL-3 release in vitro A n t i c a n c e r antibiotic bleomycin was tested over a wide non-cytotoxic dose range, but did not alter the release o f IL-3 from C o n A stimulated m o u s e splenocytes. T h e experiment was done in triplicate (see Table 1 which is representative o f three separate experiments).
Effect o f 4-OOH-cyclophosphamide on the release o f IL-3 in vitro 4 - O O H - c y c l o p h o s p h a m i d e was tested over a dose range o f 1 - 100 n g / m l , but n o statistically
Effect of Anticancer Drugs
1181
251
2O o?"
-,2 _zx15 i:,-o z~ I,-,,,,lO
JE
5
Medium only
NRS
anti-lL-3
rlL-3
IL-3
IL-3 sup IL-3 sup + NRS + anti-lL-3
60u / ml Fig. 5. Effect of anti-IL-3 serum on the activity of supernatants in the AC2 cell bioassay. The results are mean counts/rain of triplicate samples along with S.D. at half dilution of samples in the assay. AC2 cells in medium gave counts/rain x 10 ~ = 266 ± 36, AC2 cells in 1 : 40 anti-IL-3 serum gave 233 + 76 and AC2 cells in NRS gave a reading of 678 ± 89.
Table 1. Effect of bleomycin on IL-3 release in vitro Bleomycin (ng/ml)
[3HI Thymidine uptake (mean counts/rain _+ S.D.), dilution of supernatant 1/2
0 1 10 100
10210 10920 11603 10323
± ± ± ±
1/4 560 530 744 1093
8257 8303 8740 7677
1/8
± 499 ± 310 _+ 537 ___ 1419
6550 6183 6737 7156
± + ± ±
1/16 1478 334 442 1146
4327 4970 5013 4607
± 345 ± 459 _+ 130 ± 770
1 x 10~ mouse spleen cells/ml were incubated with Con A 0.5 tag/ml and various concentrations of bleomycin for 24 h. Supernatants were dialysed to remove the drug and IL-3 activity assayed in AC2 cell bioassay. The results are given as [rH] thymidine uptake. (Counts/rain) using mean of three samples of dilutions with standard deviations. Negative control of AC2 cells in medium only gave counts/rain 1083 ± 291 and 25 u / m l rmu IL-3 gave counts/min 10533 = 224 in the same assay.
Table 2. Effect of 4-bydroperoxycyclophosphamide on 1L-3 release in vitro
[3H] Thymidine uptake (mean counts/min ± S.D.),
4-hp-cyclophosphamide (ng/ml)
dilution of supernatant 1/2
0 1 10 100
9717 10808 11659 9552
± ± ~ ±
1/4 331 563 934 312
7780 8075 8819 8235
± ± ± ±
1/8 788 600 676 791
5021 5870 5861 5248
± ± ± ±
1/16 662 763 634 357
3294 4509 3909 4132
± ± ± ±
715 798 540 567
1 x l0 n mouse spleen cells/ml incubated with Con A 0.5 t~g/ml and various concentrations of 4-hydroperoxycyclophosphamide for 24 h. Supernatants were dialysed to remove the drug and IL-3 activity assayed in A C 2 cell bioassay. The results are given as mean counts/min of triplicate samples of dilutions with S.D. Negative control of AC2 cells in medium only gave counts/rain 1494 _+ 85 and 25 u / m l rmu IL-3 gave counts/rain 11227 _+ 2943 in the same assay.
S. 1. HASAN and J. L. TURK
1182
Table 3. Effect of FK156 on IL-3 release in vitro FK156 (ng/ml)
[3H] Thymidine uptake (mean c o u n t s / m i n ± S.D.), dilution of supernatant 1/2
0 1 10 100
15389 15361 16169 14375
± ± ± ±
1/4 951 742 722 1619
14541 14766 15587 15675
± ± ± ±
1/8 756 644 254 1460
11212 10713 11318 11287
± ± ± ±
1/16 305 751 204 167
7635 6767 7455 7186
± ± ± ±
970 497 715 166
1 x 10~ murine splenocytes/ml were incubated with Con A 0.5 tag/ml and concentrations of FK156 for 24 h. Supernatants were dialysed to remove the drug and IL-3 activity assayed in AC2 cell bioassay. The results are given as mean counts/rain of triplicate samples of dilutions along with S.D. Negative control of AC2 cells in medium only gave counts/rain 864 ± 133 and 25 u / m l r m u 1L-3 gave c o u n t s / m i n 19181 ± 3297 in the same assay. Table 4. Effect of FK565 on 1L-3 release in vitro FK565 (ng/ml)
[~H] Thymidine uptake (mean c o u n t s / m i n ± S.D.), dilution of superuatant 1/2
0 1 10 100
6749 6499 6021 6395
± ± ± ±
1/4 611 256 412 94
5340 5093 5134 5390
± ± ± ±
1/8 307 204 55 272
4463 3609 3916 3875
± ± ± ±
1/16 704 133 700 79
2739 2819 2997 2837
± ± ± ±
120 500 10 557
1 )< 106 m o u s e splenocytes/ml were incubated with C o n A 0.5 # g / m l and concentrations of FK565 for 24 h. Supernatants were dialysed to remove the drug and IL-3 activity assayed in AC2 cell bioassay. The results are given as mean counts/rain of triplicate samples of dilutions along with S.D. Negative control of AC2 cells in medium only gave c o u n t s / m i n 876 ± 95 and 25 u / m l rmu IL-3 gave c o u n t s / m i n 13210 ± 1050 in the same assay. Table 5. Cytotoxicity of Adriamycin and vincristine on Con A stimulated murine spleen cells
Effect o f FK565 and FKI56 on the release o f lL-3 in
Drug concentration (per ml)
F K 5 6 5 a n d F K I 5 6 a r e low m o l e c u l a r w e i g h t peptides with antimicrobial and anticancer activity. F K 5 6 5 is a s y n t h e t i c a n a l o g u e o f F K I 5 6 . F K I 5 6 is i s o l a t e d f r o m c u l t u r e s o f Streptomyces violaceus. B o t h t h e s e p e p t i d e s h a v e b e e n r e p o r t e d to p o s s e s s immunomodulatory actions (Mine, Watanabe, T a w a r a , Y o k o t a & N i s h i d a , 1983; P a l a c i o s , 1985). FK565 and FKI56 were tested for their effect on C o n A i n d u c e d IL-3 r e l e a s e f r o m m o u s e s p l e n o c y t e s , b u t d i d n o t alter t h e release o f IL-3 (see T a b l e s 3 a n d 4). A series o f t h r e e e x p e r i m e n t s w a s c a r r i e d o u t f o r b o t h d r u g s to c o n f i r m t h e r e s u l t s . T h e a p p a r e n t i n h i b i t i o n o f IL-3 b y A d r i a m y c i n and vincristine could perhaps be the effect of u n d i a l y s e d d r u g in t h e s u p e r n a t a n t i n h i b i t i n g t h e A C 2 cells in t h e IL-3 b i o a s s a y . T o e x c l u d e t h i s possibility the following experiment was performed. One drug-free control supernatant was divided into t h r e e a l i q u o t s . A d r i a m y c i n at 100 n g / m l w a s a d d e d to o n e p o r t i o n a n d v i n c r i s t i n e at t h e s a m e c o n c e n t r a t i o n to t h e s e c o n d , w h i l s t t h e t h i r d p o r t i o n r e m a i n e d u n t r e a t e d . All t h r e e s u p e r n a t a n t s were d i a l y s e d a n d a s s a y e d as n o r m a l a n d s h o w e d t h e s a m e
Zero 1 ng 10 ng 100 ng I #g
% Viable cell recovery _ standard variations Adriamycin
Vincristine
82.5 84.0 81.0 77.6 33.0
82.5 80.0 80.0 82.0 45.3
__. 7 ___6.6 ± 7.3 ± 8 ± 5
___7 ± 7 _ 6.3 ± 10 ± 3.5
1 × l& mouse splenocytes were incubated with 0.5 tag/ml Con A and Adriamycin or vincristine for 24 h. The cells were then collected and viable cells counted using trypan blue, and the percentage of cell recovery calculated. The results are means of three experiments together with standard deviations.
significant effect (P<0.02) was observed on the release of IL-3 from Con A stimulated murine spleen cell c u l t u r e s (see T a b l e 2). A series o f t h r e e e x p e r i m e n t s w a s c o m p l e t e d to c o n f i r m t h e r e s u l t a n d n o e f f e c t w a s n o t e d in a n y e x p e r i m e n t .
vitro
Effect of Anticancer Drugs IL-3 activity on AC2 cells, confirming that Adriamycin and vincristine at 100 ng/ml are effectively removed by the dialysis procedure.
DISCUSSION This study is part of our investigation of the modulation of cytokines by anticancer drugs in which we have previously reported the effect of anticancer drugs on the release of IL-1, IL-2, interferon},, (IFN},) and TNFa (Ahmed, Hamied & Turk, 1987; Ahmed & Turk, 1989; Ahmed, Vander Meide & Turk, 1989; Hamied, Parker & Turk, 1986; Hamied, Parker & Turk, 1987; Hamied & Turk, 1987; Hasan, Ahmed & Turk, 1990). We have now extended the work to include the haematopoietic cytokine IL-3. In the present study, the anticancer antibiotic Adriamycin was found to inhibit the release of IL-3 from Con A stimulated murine splenocytes at noncytotoxic concentrations. It is interesting to observe that Adriamycin enhances the release of IL-I and especially IL-2 (Hamied et al., 1987; Hamied & Turk, 1987; Ehrke, Cohen & Mihich, 1987). There are, however, other reports of this drug inhibiting the release of migration - inhibition-factor (MIF) as reported by Mantovani (1982). Moreover, we have reported that Adriamycin did not affect the release of IFNy (Ahmed et al., 1989). The other anticancer antibiotic, bleomycin, was tested and it failed to exert any effect on the release of IL-3 in our experiments. Although bleomycin is known to enhance the release of IL-1 and IL-2 (Hamied et al., 1986, Hamied & Turk, 1987) it did not affect the release of IFN}, (Ahmed et al., 1989). In clinical usage Adriamycin causes severe myelosuppression, resulting in leukopenia, thrombocytopenia and anaemia, whilst in comparison with other antineoplastic agents, bleomycin causes minimal bone marrow toxicity (Gilman, Goodman & Gilman, 1980). The severe myelosuppression caused by Adriamycin in patients could perhaps be attributed partly to the inhibition of IL-3 by the drug. The differential effects of Adriamycin and bleomycin on haemopoietic functions indicate that these drugs have a highly selective immunomodulating effect. The anticancer drug vincristine, which is an alkaloid derived from the periwinkle plant, resulted in inhibition of the release of IL-3 at non-cytotoxic concentrations. Vincristine has previously been shown to inhibit the release of IL-2 and interferon}, (Ahmed et al., 1987, 1989) which, like IL-3, are products mainly of T-cells. These findings are
1183
corroborated by the observation that vincristine suppresses various T-cell responses (Borel, 1976; Ryoyama, Mace, Ehrke & Mihich, 1982). Cyclophosphamide, which is used for the treatment of leukaemias, lymphomas, multiple myeloma, as well as other turnouts, and in graft-vshost disease has been shown to exert a wide range of immunomodulatory effects. The in vitro active analogue 4-OOH-cyclophosphamide was tested, but it did not alter the release of IL-3 in splenocyte cultures. Previous work has shown that 4-OOHcyclophosphamide at non-cytotoxic levels inhibits the release of IL-2 (Hamied et al., 1987) but does not affect the release of IFN}' (Ahmed et al., 1989) from splenocytes in vitro. Moreover, 4-OOHcyclophosphamide did not alter the release of IL-1 and TNF from macrophages (Hamied et al., 1987; Hasan et al., 1990). In another work, Kaufman, Metelemann, Diamanstein & Hahn (1984) have shown that 4-OOH-cyclophosphamide did not affect the release of IL-1 or IL-2 in vitro from Listeria monocytogenes immune T-cells up to a concentration of 3 tag/ml. It is notable that amongst all the cytokines tested in our studies (IL-I, IL-2, TNFcr, IFNy and IL-3) only IL-2 was modulated by 4-OOH-cyclophosphamide and its release suppressed. It is likely, therefore, that the immunosuppressive effects of cyclophosphamide are mediated mainly by mechanisms other than cytokine modulation. In the present study, we tested the experimental anticancer and immunomodulatory peptides FK565 and FK156 for their effect on IL-3 release. These peptides did not alter the release of IL-3 from spleen cell cultures. In previous work we have reported enhancement of IL-1 by FK565 and FKI56 (Ahmed & Turk, 1989). In that same work, suppression of IL2 release has been reported from spleen cell cultures in vitro. FK565 has been shown to cause macrophage activation in vivo (Watanabe, Tawara, Mine & Kikuchi, 1985) and to enhance the release of TNFa from macrophages in vitro (Hasan et al., 1990). In a further study, these peptides did not alter the release of IFNy (Ahmed et al., 1989). These findings would seem to point to the possibility that these immunoactive anticancer peptides are selective in their action on the various cytokines. Cyclosporin A, an immunosuppressive agent which has been reported to inhibit the release of IL-3 splenocytes (Palacios, 1985), was included in this investigation to confirm the results of these experiments. Cyclosporin A, when added to murine spleen cell cultures stimulated with suboptimal concentrations of Con A, decreased the release of
1184
S. 1. HASAN and J. L. TURK
IL-3 in the supernatant. It is interesting to note that cyclosporin A has an established place in the prevention and treatment o f graft-vs-host disease which is a condition k n o w n to be associated with elevation o f serum IL-3 levels in experimental animals ( C r a p p e r & Schrader, 1986). The present findings may contribute to a better u n d e r s t a n d i n g o f the m o d e o f action o f anticancer drugs, and o f the pathophysiological role o f IL-3. The finding o f suppression o f IL-3 by A d r i a m y c i n
and vincristine is i m p o r t a n t , as IL-3 exerts some control on haemopoiesis and immunity. These drugs, in therapeutic doses, exert toxic effects on these functions. Acknowledgements - - The funding for this work was provided by the Ministry of Science and Technology of the Government of Pakistan. The authors are grateful to Dr John Garland for the gift of cell lines and helpful advice. We also thank Mrs June Saxby for typing the manuscript.
REFERENCES
AHMED, K., HAMIED, T. A. A. & TURK, J. L. (1987). Inhibition of interleukin-2 by vincristine and vinblastine. lmmunopharmac, lmmunotoxic., 9, 391 - 407. AHMED, K. & TURK, J. L. (1989). Effect of anticancer agents neothramycin, aclacinomycin, FK565 and FK156 on the release of interleukin-2 and interleukin-1 in vitro. Cancer Immun. Immunother., 28, 8 7 - 92. AHMED, K., VAN DER ME1DE, P. H. & TURK, J. L. (1989). Effect of anticancer drugs on the release of interferon), in vitro. Cancer Immun. Immunother., 30, 213-218. BOREL, J. F. (1976). Comparative study of in vitro and in vivo drug effects on cell-mediated cytotoxicity. Immunology, 31, 631-641. COLV1N, M., PADGETT, C. A. & FENSELOU,C. (1973). A biologically active metabolite of cyclophosphamide. Cancer Res., 33, 915-918. CRAPPER, R. M. & SCHRADER,J. W. (1986). Evidence for the in vivo production and release into the serum of a T cell lymphokine, persisting-cell stimulating factor (PSF), during graft-versus-host reactions. Immunology, 57, 553 - 558. CRAPPER, R. M., VA1RO, G., HAMILTON~ J. A.~ LEWIS, I. C. & SCHRADER, J. W. (1985). Stimulation of bone marrowderived and peritoneal macrophages by a T lymphocyte-derived hemopoietic growth factor, persisting cell-stimulating factor. Blood, 66, 859-866. DAVIGNON, J. L., KIMOTO, M., KINDLER, V., DE KOSSODO, S., VASSALLI,P. & IZU1, S. (1988). Selective production of interleukin 3 (IL-3) and granulocyte - macrophage colony-stimulating factor (GM-CSF) in vitro by murine L3T4 ~ T cells: lack of spontaneous IL-3 and GM-CSF production by Ly-2-/L3T4-Ipr subset. Eur. J. Immun., 18, 1367- 1372. EHRKE, M. J., COHEN, S. A. & MIHIC'H, E. (1982). Selective effects of Adriamycin on murine host defence systems. Immunology, 65, 5 5 - 77. GAUCHAT, D., GAUCHAT, J. F., BETTENS, F., DE WECK, A. L. & STADLER, B. M. (1990). Cytokine gene expression in atopics: effect of IL-4 on IL-I/3 and IL-6 mRNA levels. Eur. Cytokine Nt., 1, 85-90. GILMAN, A. G., GOODMAN, L. S. & GILMAN, A. (1980). The Pharmacological Basis o f Therapeutics, 6th Edn, pp. 1293 - 1295. Macmillan, New York. HAMIED, T. A. A., PARKER, D. & TURK, J. L. (1986). Potentiation of release of interleukin 2 by bleomycin. Immunopharmacology, 12, 127- 134. HAMIED, T. A. A., PARKER,D. & TURK, J. L. (1987). Effect of Adriamycin, 4-hydroperoxycyclophosphamideand ASTAZ 7557 (Inn Mafosfamide) on the release of IL-I and IL-2 in vitro. Int. J. Immunopharmac., 9, 355-361. HAMIED, T. A. A. & TURK, J. L. (1987). Enhancement of interleukin 2 release in rats by treatment with bleomycin and Adriamycin in vivo. Cancer Immun. lmmunother., 25, 245-249, HAPEL, A. J. & YOUNG, I. G. (1987). Abnormal expression of interleukin 3 and leukaemia. Anticancer Res., 7, 661 -668. HAPEL, A. J. & YOUNG, I. Q. (1988). Molecular biology of interleukin-3. Lymphokines, (ed. Schrader, J. W.) Vol. 15, pp. 9 1 - 26. Academic Press, New York. HASAN, S. I., AHMED, K. & TURK, J. L. (1990). Effect of anticancer drugs on the release of tumour necrosis factors in vitro. Cancer Immun. Immunother., 30, 363 - 366. HIRASAWA, N., OHUCH1, K., KAWARASAKI,K., WATANABE, M. & TSURUFIJI, S. (1989). Occurrence of histamineproduction-increasing factor in postanaphylactic phase of allergic inflammation. Int. Arch. Allergy appl. Immun., 88, 386- 393.
Effect of Anticancer Drugs
1185
KAUFMAN, S. H. E., METELEMANN, C., DIAMANSTEIN, T. & HAHN, H. (1984). Resistance to 4hydroperoxycyclophosphamideof T cells involved in cell mediated antibacterial immunity. Int. J. lmmunopharmac., 6, 8 1 - 8 5 . MANTOVANI,A. (1982). Advances in Pharmacology and Chemotherapy, the Interaction o f Cancer Chemotherapy Agents with Mononuclear Phagocytes, Vol. 19, pp. 35-66. Academic Press, New York. METCALF, D. (1987). The molecular control of normal and leukaemic granulocytes and macrophages. Proc. R. Soc. Lond., B230, 389- 423. MINE, Y. WATANABE, Y., TAWARA, S., YOKOTA, Y. & N1SHIDA, M. (1983), lmmunoactive peptides FKI56 and FK565; enhancement of host defence mechanisms against infection. ]. Antibiot. (Tokyo), 36, 1059- 1066. PALA(IOS, R. (1985). Cyclosporin A inhibits antigen and leclin-induced but not constitutive production of interleukin 3. Eur. J. Immun., 15, 204-206. PALACIOS, R. & GARLAND, J. (1984). Distinct mechanisms may account for the growth-promoting activity of interleukin 3 on cells of lymphoid and myeloid origin. Proc. natn. Acad. Sci. U.S.A., 81, 1208- 1211. PEBtJS,:.)UE, M. J., FAY, C., LAVAC;E, M., SEMPERE, C., SAELAND, S., CAtJX, C. & MANNONI, P. (1989). Recombinant human IL-3 and G-CSF act synergistically in stimulating the growth acute myeloid leukemia cells. Leukemia, 3, 200 - 205. RVOVAMA, K., MACE, K., EHRKE, M. J. & MrHICH, E. (1982). The differential sensitivities of T cell immune functions to vincristine and vinblastine. Int. J. Immunopharmac., 4, 187- 193. VERALA, A., VIDALLER,A., LLORENTE, L. & SEGOVlA, J. D. A. (1988). Presence of an IL-3-producing suppressor T cell resistant to cyclosporin A in the peripheral blood of patients with systemic lupus erythematosus. Clin. exp. lmmun., 73, 424 - 429. WATANABI2, Y., TAWARA,S., M~NE, Y. & KIKUCHI, H. (1985). Immunoactive peptides FKI56 and FK565 - - activation of mouse macrophages. J. Antibiot. (Tokyo), 38, 1781.