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Immunopharmacological study of CCA(Lobenzarit disodium), an anti-arthritis agent — I. Abrogation of IL 1 secretion by LPS-stimulated human monocytes and induction of γ-interferon production with CCA
lnt. J. lmmunopharmac., Vol. 8, No. 3, pp. 323-328, 1986. Printed in Great Britain.
0192-0561/86 $3.00+ .00 © 1986 International Society for Immunopharmacology.
I M M U N O P H A R M A C O L O G I C A L S T U D Y OF C C A ( L O B E N Z A R I T D I S O D I U M ) , A N A N T I - A R T H R I T I S A G E N T - - I. A B R O G A T I O N OF IL 1 S E C R E T I O N BY LPS- S T I M U L A T E D H U M A N M O N O C Y T E S A N D I N D U C T I O N OF y - I N T E R F E R O N P R O D U C T I O N W I T H C C A MASAO FUJIMOTO, ISAMU SUGAWARA,MAKOTO KIMOTO, SHIGEAKI ISHIZAKAand TADASU TSUJll Third Department of Internal Medicine, Nara Medical University, 840 Shijo-cho, Kashihara City, Nara 634, Japan (Received 23 January 1985 and in final form 25 September 1985)
Abstract - - In vitro effects of CCA, an anti-arthritis agent, were studied upon autologous mixed lymphocyte
reaction (AMLR), lymphocyte mitogenesis, IL 1 and IL 2 production, immunoglobulin production and y-interferon (IFN) production. CCA at 50 /ag/ml, which was not toxic to cells, blocked AMLR, IL 1 production and immunoglobulin production (IgM and IgG) significantly, while CCA at the same dose did not affect IL 2 production and lymphocyte mitogenic responses to Staphylococcus aureus Cowan I(SAC) and pokeweed mitogen(PWM). CCA at both 20 ng/ml and 20/ag/ml induced human y/IFN. Addition of IL 1 and/or IL 2 reversed inhibitory effect of CCA on AMLR. These data suggest that CCA exerts its actions by mainly affecting T cells and monocytes and can be used as an immunomodulator.
CCA(Lobenzarit disodium), whose generic name is disodium 4-chloro-2,2'-iminodibenzoate, developed in Japan is therapeutically effective in experimental arthritis in rats (Ohsugi, Hata, Tanemura, Nakano, Matsuno, Takagaki, Nishii & Shindo, 1977), spontaneous autoimmune disorders in MRL/1 mice (Abe, Shiokawa, Ohishi, Hata & Takagaki, 1981) and autoimmune kidney disease in N Z B / N Z W F1 hybrid mice (Ohsugi, Nakano, Hata, Niki, Matsuno, Nishi & Takagaki, 1978). CCA is now being investigated as a probable chemotherapeutic agent for rheumatoid arthritis (RA) in humans. However, the immunological study of CCA has not been fully performed. It has, so far, been reported that NK activity of human mononuclear cells was significantly increased after incubation with 10 ng/ml of CCA and the expressions of the receptors for IgG-Fc(F,-R) on human lymphocytes were augmented by CCA (Itoh, Saito, Kumagai & Kosaka, 1981). The present study was undertaken to investigate the effects of CCA on human immunocompetent cells in vitro, using various assay systems including plaque forming cell (PFC) assay, autologous mixed
lymphocyte reaction (AMLR), IL 1 and IL 2 activity, y-interferon (IFN) production and lymphocyte mitogenic responses. We report here that CCA exerted its actions by blocking IL 1 production by human monocytes and inducing y-IFN production and can be used as an immunomodulator.
EXPERIMENTAL PROCEDURES
Human mononuclear cell (MNC) preparation Heparinized blood was obtained from healthy donors 2 0 - 6 0 yr old. MNC were separated by F i c o l l - C o n r a y 400 flotation method (Sugawara, Ishizaka, Tsujii & De Ley, 1984). The number and percentage of viable cells was determined by trypan blue dye exclusion. Reagents CCA(Lobenzarit disodium) was kindly provided by Chugai Pharmaceutical Co., Ltd., Tokyo, Japan. LPS (Difco Laboratories), Con A (Sigma Chemical
Please address correspondence to: Dr. Isamu Sugawara, Department of Basic Research, Research & Development Laboratories, Hoechst Japan Ltd., 1-3-2 Minamidai, Kawagoe, City, Saitama 350, Japan 323
324
Co.,
MASAO FUJIMOTO el al.
Ltd.),
PWM
(P-L
Laboratories)
and
Staphylococcus aureus Cowan I (SAC, Wako Pure Chemical Co., Tokyo) were used as mitogens. Mitomycin C was purchased from Sigma Chemical Co., Ltd.. Human ultrapure interleukin 1 and 2 (GUPI-1 and GUPI-2) were obtained from Genzyme corporation, U.S.A. Rabbit anti-human y-IFN antibody and y-IFN were kindly supplied by Dr. M. De Ley, Rega Institute, Belgium.
Autologous mixed lymphocyte reaction (AMLR) MNC (2 × 10S/well) as responders and mitomycin C (40 /ag/ml)-treated MNC (2 × 10S/well) as stimulators were incubated in the presence or absence of CCA in flat-bottomed microplates at 37°C for 7 days. In some experiments, appropriate amounts of interleukin 1 and 2 were added to the cultures. Cellular proliferation was determined by 3H-thymidine uptake (0.25 KBq/well; specific activity, 185 GBq; New England Nuclear) during the last 6 h of the culture period. The cells were harvested with a semi-automated cell harvester and radioactivity was counted in a liquid scintillation spectrometer.
IL 1 production Plastic adherent cells were cultured in RPMI 1640 with 1% FCS in the presence of LPS (50tag/ml) a n d / or CCA (50 tag/ml) at 37°C for 24 h in a CO2 incubator. The culture fluid was obtained by centrifugation (500 g at 4°C for 25 min).
IL 1 assay Thymocytes from C3H/HeJ mice were used as the target cells. The cells in RPMI 1640 with 5% FCS were incubated in 96-well microculture plates for 72 h at 37°C in a 5% CO2 incubator. IL 1 containing samples were tested in triplicate by the addition of 50 /al of sample per thymocyte culture (Sugawara, Ishizaka & M611er, 1982).
IL 2 production IL 2 containing supernatants were obtained by incubating MNC (2 × 106/ml) in plastic tubes (Falcon plastics; 2058) with Con A (2/ag/ml) and/or CCA (50 tag/ml) for 24 h.
IL 2 assay The T cell growth stimulatory activity of IL 2 was assayed, using IL 2 dependent T cells derived from a PHA-initiated T cell line (Palacios, Sugawara &
Fernandez, 1982b). The cultures containing 50 tal of IL 2 containing supernatants received 1 x 20" viable IL 2 dependent T cells. Cell proliferation was determined by 3H-thymidine uptake during the last 6 h of a 2 day culture period performed at 37°C.
Lymphocyte blastogenic assay MNC (5 x 10S/well) in 200/al of RPMI 1640 with 5°70 FCS were incubated in the presence of PWM (50 ~g/ml) or SAC (10-Sv/v) at 37°C for 4 days in a CO2 incubator. Tritiated thymidine was introduced 6 h before the termination of the culture. Radioactivity was counted in a liquid scintillation spectrometer.
Plaque forming cell (PFC) assay MNC (5 × 10S/well) were cultured in the presence of PWM (50/ag/ml) with or without CCA (20/ag/ml or 50 /ag/ml) for 7 days. PFC was evaluated by means of protein A-SRBC hemolytic plaque assay (Gronowizc, Coutinho & Melchers, 1976). Briefly, 25/A of the cultured cell suspension (5 × l&/ml) was added to 0.2 ml of 0.5% liquid agar with 3% DEAE dextran containing 25 /al of a protein A-coupled SRBC (dilution: 1: 8). Rabbit anti-mouse IgM or IgG (25/A, diluted 1/5) were added to cells in tubes. The above mixture was placed on petri dishes, covered with cover glasses and incubated at 37°C for 4 h. The results were expressed as PFC per 10s viable cells. All the experiments were done in duplicate.
Effect o f CCA on y-interferon production Human y-interferon production was evaluated by PFC assay with some modifications (Gronowicz et al., 1976; Palacios, Martinez-Maza & De Ley, 1983; Sugawara et al., 1984). Rabbit antihuman yinterferon antibody (final dilution: 1:120) was used instead of rabbit antihuman IgM antibody. The results were expressed as y-IFN PFC/106 viable cells. All the assays were conducted using a final dilution of anti human y-IFN antibody of 1:120, since we found this dilution to be adequate for the development of y-IFN PFC.
Statistical analysis Statistical analyses were made using Student's t-test.
RESULTS
Kinetic study o f the inhibitory effect o f CCA on AMLR One-way stimulated AMLR was performed in the presence or absence of various doses of CCA at 37°C
325
I m m u n o p h a r m a c o l o g i c a l study of C C A Table 1. Kinetic study of dose-dependent effect of C C A on AMLR*
Table 3. Effect of C C A on IL 1 production by LPSstimulated h u m a n monocytes*
Addition of C C A
Supernatant source* (dilution: 1:4)
-
Mean ± S.D. (counts/min) 29443 29507 33663 32330 28850 29113 36388 28191 5684 172
-
20 n g / m l 50 n g / m l 100 n g / m l 500 n g / m l 1 Mg/ml 10 Mg/ml 20 Mg/ml 40 Mg/ml 50 Mg/ml
± ± ± ± ± ± ± ± ± ±
442t 3061 801 805 522 1091 3395 2335 372* 12t
*Autologous mixed lymphocyte reaction (AMLR) was performed in the presence or absence of various doses of C C A at 37°C for 7 days. Cellular proliferation was determined by 3H-thymidine uptake during the last 6 h of the culture period. The results express m e a n ± S.D., each in quadruplicate. *Significantly different from control (P<0.01). f o r 7 d a y s . A s s h o w n in T a b l e 1, 50 / a g / m l C C A inhibited AMLR s i g n i f i c a n t l y , w h i l e 20 M g / m l t h r o u g h 10 M g / m l C C A d i d n o t i n h i b i t it. C C A o f 50 M g / m l w a s n o t c y t o t o x i c to h u m a n M N C .
Effect o f addition o f human purified IL 1 and~or IL 2 upon A M L R M N C (2 × 105/well) a n d m i t o m y c i n C (40 M g / m l ) t r e a t e d M N C (2 x 1 0 V w e l l ) w e r e c o c u l t u r e d in t h e p r e s e n c e o r a b s e n c e o f C C A (50 M g / m l ) at 3 7 ° C f o r 7 days. At the same time, the culture was s u p p l e m e n t e d w i t h 5 / A o f h u m a n I L 1 a n d / o r 10/al
Con A (1 Mg/ml)
-C C A (50 Mg/ml) LPS (50 Mg/ml) LPS (50 ~g/ml) and C C A (50 Mg/ml) -
-
C C A (50 ~g/ml) LPS LPS and C C A 5 MI of pure h u m a n IL 5 M1 of pure h u m a n IL and C C A (50/ag/ml) 5 MI of pure h u m a n IL 5/al of pure h u m a n IL and C C A (50 Mg/ml)
1 1 1 1
-
Activity (counts/min) 269 ± 7 281 _ 3 1670 ± 14
+ + + + -
321 6841 7998 24210 26703 3106
± 2 ± 64 + 432 ± 2280 _+ 920 ± 140
+
2932 ± 43 46520 ± 4300
+
46446 _+ 6041
* C 3 H / H e J murine thymocytes (106/well) were incubated in the presence or absence of IL 1 or IL 1 containing supernatant at 37°C for 3 days. Cellular proliferation was evaluated by 3H-thymidine uptake during the last 6 h of the culture period. All the data show m e a n ± S.D., each in quadruplicate. *Plastic adherent mononuclear cells were cultured with LPS (50 Mg/ml) in the presence or absence o f C C A (50/ag/ml) at 37C for one day. Thereafter, the culture fluid was collected by centrifugation at 500 g at 4°C for 25 min. The culture supernatants collected were dialyzed against double distilled water for 3 days. *Significantly different from control at P<0.01.
o f I L 2 to see w h e t h e r I L l a n d / o r I L 2 c a n o v e r c o m e t h e i n h i b i t o r y e f f e c t o f C C A o n A M L R . C C A (50 Mg/ml) b l o c k e d AMLR significantly but its
Table 2. Effect of IL 1 a n d / o r IL 2 upon depressed A M L R induced by CCA* AMLR
ILl
IL2
CCA
Activity (counts/min)
+ + + + + + + +
+ + + +
+ + + +
+ + + + -
7725 ± 139* 2119 ± 184* 9034 ± 475t 11315 ± 233t 14000± 205t 12367 ± 786 11005 ± 535 16935 ± 533
*MNC (2 × 10S/well) and mitomycin C (40 ~g/ml)-treated M N C (2 × 10S/well) were incubated in the presence or absence of C C A in flat-bottomed microculture plates at 37°C for 7 days. The cultures were supplemented with IL 1 (5 Ml) a n d / o r IL 2 (10 Ml) and incubated at 37°C for 7 days. Cell proliferation was determined by 3H-thymidine uptake during the last 6 h o f the culture period. Control values for mitomycin C-treated M N C and untreated M N C alone were 1670 _+ l l 0 (counts/min) and 2538 ± 224 (counts/min), respectively. *Statistically significant compared with control group (,o
Table 4. Effect o f C C A on IL 2 production by C o n Astimulated T cells* Supernatant source (dilution: 1:4) -M N C alone C C A (50 ~g/ml) Con A (2 Mg/ml) 20ul of h u m a n pure IL 2 10ul of h u m a n pure IL 2 20ul of h u m a n pure IL2 and C C A (50/~g/ml) C o n A and C C A
Activity (counts/min) 2341 2496 3771 8245 10933 8932
± 56* ± 94* ± 46 _+ 678 t ± 12 ± 241
8845 ± 2 7984 ± 37t
*One milliliter of 2 x l0 s h u m a n M N C were cultured with C o n A (2 Mg/ml) in the presence or absence o f C C A (50/ag/ ml) at 37°C for one day. The supernatants were collected and were dialyzed against double distilled water for 3 days and their IL 2 activity was assessed in a microassay based upon the IL 2-dependent proliferation of a PHA-initiated T cell line (3 x 103/well). *Significantly different from control (P<0.01).
326
MASAO FUJIMOTO et al. Table 5. Effect of C C A on D N A synthetic responses induced by P W M or SAC* M N C (5 x 10S/well)
SAC (10-3v/v)
P W M (50/ag/ml)
+ + -
+ +
1562 33387 8747 2050 29022 11295
+ 53 +_ 3665* + 91' _+ 56 _+ 613' +_ 191"
+ + -
+ +
2283 27819 14313 1980 32831 13000
_+ 114 + 416' + 117" _+ 33 +_ 271' _+ 278*
Experiment 1 + + + + CCA(50 ~g/ml) + CCA(50/ag/ml) + CCA(50/ag/ml) Experiment 2 + + + + CCA(50/ag/ml) + CCA(50/ag/ml) + CCA(50 gg/ml)
Activity (counts/min)
*MNC (5 × 10S/well) were cultured for 4 days with or without SAC, P W M a n d / o r CCA. D N A synthesis was evaluated by 3H-thymidine uptake during the last 6 h of the incubation period. All the values show m e a n _+ S.D., each in quadruplicate. *Not significant compared with control at P<0.01. *Not significant compared with control at P<0.001.
i n h i b i t o r y e f f e c t o n A M L R c o u l d be o v e r c o m e at a d d i t i o n o f h u m a n I L 1 a n d / o r I L 2 ( T a b l e 2).
Effect o f CCA on IL 1 generation C 3 H / H e J m u r i n e t h y m o c y t e s (1 x l & / w e l l ) were i n c u b a t e d in t h e p r e s e n c e o r a b s e n c e o f purified IL 1 or I L 1 c o n t a i n i n g s u p e r n a t a n t s f r o m v a r i o u s sources. C C A ( 5 0 / a g / m l ) a b r o g a t e d g e n e r a t i o n o f IL 1 b y L P S s t i m u l a t e d h u m a n m o n o c y t e s s i g n i f i c a n t l y (Table 3).
P H A - i n i t i a t e d T cell line. CCA d i d n o t b l o c k p r o d u c t i o n o f I L 2 b y h u m a n l y m p h o c y t e s ( T a b l e 4).
Effect o f CCA upon DNA synthesis induced by P W M or S A C We examined whether CCA blocked DNA s y n t h e t i c r e s p o n s e s to P W M , T cell d e p e n d e n t B cell m i t o g e n a n d S A C , T cell i n d e p e n d e n t B cell m i t o g e n . A s d e s c r i b e d in T a b l e 5, C C A (50 # g / m l ) d i d n o t inhibit DNA synthesis by PWM or SAC.
Effect o f CCA on IL 2 production O n e milliliter o f 2 x 10 ~ h u m a n M N C w e r e c u l t u r e d in t h e p r e s e n c e o r a b s e n c e o f C C A ( 5 0 / a g / m l ) at 3 7 ° C f o r 24 h . T h e s u p e r n a t a n t s w e r e c o l l e c t e d a n d t h e i r I L 2 a c t i v i t y w a s a s s e s s e d in a m i c r o a s s a y based upon the IL 2 dependent proliferation of a
Effect o f CCA on P WM-driven B-cell differentiation S i n c e we f o u n d t h a t C C A b l o c k e d I L 1 p r o d u c t i o n and speculated that CCA blocked B-cell d i f f e r e n t i a t i o n , we i n v e s t i g a t e d t h e e f f e c t o f C C A o n
Table 6. Effect of C C A upon immunoglobulin plaque forming cells (PFC)* M N C (3 × 10S/well)
P W M (50 ~g/ml)
CCA
IgM PFC/106
lgG PFC/106
+ + + + + +
+ + + -
20 g g / m l 20gg/ml 50gg/ml 50 ~g/ml
33 731' 680 40 33* 33
66 890* 753 53 53* 45
*MNC (3 × 10S/well) were cultured with or without P W M a n d / o r C C A at 37°C for 7 days. SRBC-protein A plaque assay was utilized to enumerate IgM and lgG PFC. All the experiments were done in duplicate. *Significantly different from control (P<0.01).
Immunopharmacological study of CCA Table 7. Effect of CCA on y-IFN producing cells
327
DISCUSSION
We tried to clarify the mechanism of action of C C A . We speculated that C C A had inhibitory effects on T cells and H L A - D R bearing B cells and monocytes, since C C A inhibited proliferation of T cells in A M L R significantly (Palacios & M611er, 1981). Therefore, we performed experiments aiming at investigating whether IL 1 a n d / o r IL 2 can overcome inhibitory effects o f C C A on A M L R , IL 1 or IL 2 addition could reverse depressed A M L R . Thus, we conjectured that C C A was acting on T cells a n d / o r H L A - D R bearing cells. Next, to determine the site of action of C C A we studied whether C C A blocked IL 1 a n d / o r IL 2 production. C C A did not hinder IL 2 production by T cells, but prevented stimulated m a c r o p h a g e s / monocytes from production of IL 1. H o w do we interpret the data that 50/ag/ml C C A blocked P W M - d r i v e n B cell differentiation? It is likely that P F C responses decreased due to decrease of IL 1 and y-interferon did not affect P F C responses, for 20 /ag/ml CCA, inducing y-IFN production, did not block P F C responses induced by P W M . C C A did not affect B cells, since D N A synthetic responses induced by S A C and P W M were not affected with C C A . Therefore, we conjectured from our data that C C A mainly acted on T cells and monocytes. We reported previously that hydrocortisone abrogated proliferation of T cells in A M L R by rendering the IL 2 producer T cells unresponsive to IL 1 and unable to synthesize IL 2 (Palacios & Sugawara, 1982a). In our present study C C A had two actions--induction of y-IFN production and abrogation of IL 1 production. Thus, the mechanism o f action of C C A is completely different from that o f hydrocortisone. C C A is thought to be a promising chemical as an immunomodulator.
In the reported experiments here we showed that C C A was a good in vitro inducer for y-IFN production and abrogated IL 1 production by stimulated human monocytes.
Acknowledgements -- We gratefully acknowledge Dr. M. De Ley, Rega Institute, Belgium, for providing us with human yinterferon and rabbit antihuman y-interferon antibody.
Cell assay
y-IFN PFC/106 viable cells No.1 No.2 No.3
Experiment 1" MNC 350* 210' 53* MNC + Con A(2 Cg/ml 644* 429* 266* MNC+CCA(20ng/ml) 993* 756* 523* MNC(without C' ) 1 0 0 MNC(without anti y-IFN Ab) 0 0 0 MNC + Con A(without C') 0 0 0 MNC + Con A (without antiy-IFN Ab) 0 0 0 Experiment 2* MNC 912' MNC + Con A(2/ag/ml) 3264* MNC + CCA(20/ag/ml) 3840* *Human mononuclear cells were incubated in RPMI 1640 containing 5°70 FCS with or without various chemicals indicated above for one day. Thereafter, the cells were washed and resuspended in BSS. The number of IFN-yPFC was determined by a reverse hemolytic plaque assay using a purified anti IFN-y antibody (final dilution: l:120) as developing sera. In some experiments, neither C' nor anti IFN-y antibody was added to the plaque assay. *Human mononuclear cells were cultured in culture tubes in the presence or absence of a stimulant for two days at 37°C in a CO2 incubator. *Significantly different from controls (P<0.01). P W M - d r i v e n B cell differentiation in terms of plaque forming cells (PFC). As expected, 20/ag/ml C C A did not abrogate generation of P F C , whereas 5 0 / a g / m l C C A abrogated it (Table 6). Effect o f CCA on y-IFN production We studied whether C C A induced y-interferon production in terms of y-IFN P F C . As shown in Table 7, both 20 n g / m l and 20 tag/ml C C A induced y-interferon significantly, y-IFN production by C C A was comparable with that by Con A.
REFERENCES
ABE, C., SHOIKAWA,Y., OHISHI, T., HATA, S. & TAKAGAKI,Y. (1981). The effect of N-(2-carboxyphenyl)-4chloroanthranilic acid disodium salt (CCA) on spontaneous autoimmune disorders in MRL/1 mice. Ryumachi, 21 (Suppl.), 165- 170. GRONOWlCZ, E., COUTINHO,A. & MELCHERS,F. (1976). A plaque assay for all cells secreting Ig of a given type or class. Euro.J.Immun., 6, 588- 589. ITOH, K., SAITOH,F., KtJMAGAI,K. & KOSAKA,S. (1981). Depressed natural killer activity in rheumatoid arthritis and its in vitro augmentation with interferon and N-(2-carboxyphenyl)-4-chloroanthranilic acid disodium salt (CCA), an antiarthritis agent. Ryumachi, 21 (Suppl.), 69-74.
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MASAO FUJIMOTO et al.
OHSUGI, Y., HATA, S., TANEMURA,M., NAKANO, T., MATSUNO, T., TAKAGAKI,Y., NISHII, Y. & SHINDO, M. (1977). N-(2carboxyphenyl)-4-chloroanthranilic acid disodium salt: a novel anti-arthritic agent without anti-inflammatory and immunosuppressive activities. J.Pharm.Pharmac, 29, 636-637. OHSUGI, Y., NAKANO, T., HATA, S., NIKI, R., MATSUNO, T., N1SHI1, Y. 8z TAKAGAKI, Y. (1978). N-(2-carboxyphenyl)-4chloroanthranilic acid disodium salt: prevention of autoimmune kidney disease in NZB/NZW F, hybrid mice. J.Pharm.Pharmac., 30, 126-128. PALACIOS, R. • MOLLER, G. (1981). HLA-DR antigens render resting T cells sensitive to Interleukin-2 and induce production of the growth factor in the autologous mixed lymphocyte reaction. Cell.Immun., 63, 143- 151. PALAC1OS,R. & SUGAWARA,I. (1982a). Hydrocortisone abrogates proliferation of T cells in autologous mixed lymphocyte reaction by rendering the Interleukin-2 producer T cells unresponsive to Interleukin-1 and unable to synthesize the Tcell growth factor. Scand.J.Immun., 15, 2 5 - 3 1 . PALACIOS, R., SUGAWARA,1.8,~FERNANDEZ, C. (1982b). Dextran-sulfate: a mitogen for human T lymphocytes. J. Immun., 1 2 8 , 621 - 624. PALACIOS, R . , MARTINZ-MAZA,O. 8~ DE LEY, M. (1983). Production of human immune interferon (HuIF-y) studied at the single cell level. Origin, evidence for spontaneous secretion and effect of cyclosporin A. Eur.J. Immun., 13, 221 - 225. SUGAWARA, l., ISH1ZAKA,S. 8~ MOLLER, G. (1982). Carrageenans, highly sulfated polysaccharides and macrophage-toxic agents: newly found human T lymphocyte mitogens. Immunobiology, 163, 527-538. SUGAWARA, I., ISH1ZAKA,S., TsuJn, T. & DE LEY, M. (1984). The role of interferon-y (IFN-y) producing cells in clinical immunology I.OK 432, glycyrrhizin and CCA (Lobenzarit disodium) are good in vitro inducers for IFN-y production. J.Nara reed.Ass., 35, 4 2 4 - 429.
0192-0561/86 $3.00+ .00 © 1986 International Society for Immunopharmacology.
I M M U N O P H A R M A C O L O G I C A L S T U D Y OF C C A ( L O B E N Z A R I T D I S O D I U M ) , A N A N T I - A R T H R I T I S A G E N T - - I. A B R O G A T I O N OF IL 1 S E C R E T I O N BY LPS- S T I M U L A T E D H U M A N M O N O C Y T E S A N D I N D U C T I O N OF y - I N T E R F E R O N P R O D U C T I O N W I T H C C A MASAO FUJIMOTO, ISAMU SUGAWARA,MAKOTO KIMOTO, SHIGEAKI ISHIZAKAand TADASU TSUJll Third Department of Internal Medicine, Nara Medical University, 840 Shijo-cho, Kashihara City, Nara 634, Japan (Received 23 January 1985 and in final form 25 September 1985)
Abstract - - In vitro effects of CCA, an anti-arthritis agent, were studied upon autologous mixed lymphocyte
reaction (AMLR), lymphocyte mitogenesis, IL 1 and IL 2 production, immunoglobulin production and y-interferon (IFN) production. CCA at 50 /ag/ml, which was not toxic to cells, blocked AMLR, IL 1 production and immunoglobulin production (IgM and IgG) significantly, while CCA at the same dose did not affect IL 2 production and lymphocyte mitogenic responses to Staphylococcus aureus Cowan I(SAC) and pokeweed mitogen(PWM). CCA at both 20 ng/ml and 20/ag/ml induced human y/IFN. Addition of IL 1 and/or IL 2 reversed inhibitory effect of CCA on AMLR. These data suggest that CCA exerts its actions by mainly affecting T cells and monocytes and can be used as an immunomodulator.
CCA(Lobenzarit disodium), whose generic name is disodium 4-chloro-2,2'-iminodibenzoate, developed in Japan is therapeutically effective in experimental arthritis in rats (Ohsugi, Hata, Tanemura, Nakano, Matsuno, Takagaki, Nishii & Shindo, 1977), spontaneous autoimmune disorders in MRL/1 mice (Abe, Shiokawa, Ohishi, Hata & Takagaki, 1981) and autoimmune kidney disease in N Z B / N Z W F1 hybrid mice (Ohsugi, Nakano, Hata, Niki, Matsuno, Nishi & Takagaki, 1978). CCA is now being investigated as a probable chemotherapeutic agent for rheumatoid arthritis (RA) in humans. However, the immunological study of CCA has not been fully performed. It has, so far, been reported that NK activity of human mononuclear cells was significantly increased after incubation with 10 ng/ml of CCA and the expressions of the receptors for IgG-Fc(F,-R) on human lymphocytes were augmented by CCA (Itoh, Saito, Kumagai & Kosaka, 1981). The present study was undertaken to investigate the effects of CCA on human immunocompetent cells in vitro, using various assay systems including plaque forming cell (PFC) assay, autologous mixed
lymphocyte reaction (AMLR), IL 1 and IL 2 activity, y-interferon (IFN) production and lymphocyte mitogenic responses. We report here that CCA exerted its actions by blocking IL 1 production by human monocytes and inducing y-IFN production and can be used as an immunomodulator.
EXPERIMENTAL PROCEDURES
Human mononuclear cell (MNC) preparation Heparinized blood was obtained from healthy donors 2 0 - 6 0 yr old. MNC were separated by F i c o l l - C o n r a y 400 flotation method (Sugawara, Ishizaka, Tsujii & De Ley, 1984). The number and percentage of viable cells was determined by trypan blue dye exclusion. Reagents CCA(Lobenzarit disodium) was kindly provided by Chugai Pharmaceutical Co., Ltd., Tokyo, Japan. LPS (Difco Laboratories), Con A (Sigma Chemical
Please address correspondence to: Dr. Isamu Sugawara, Department of Basic Research, Research & Development Laboratories, Hoechst Japan Ltd., 1-3-2 Minamidai, Kawagoe, City, Saitama 350, Japan 323
324
Co.,
MASAO FUJIMOTO el al.
Ltd.),
PWM
(P-L
Laboratories)
and
Staphylococcus aureus Cowan I (SAC, Wako Pure Chemical Co., Tokyo) were used as mitogens. Mitomycin C was purchased from Sigma Chemical Co., Ltd.. Human ultrapure interleukin 1 and 2 (GUPI-1 and GUPI-2) were obtained from Genzyme corporation, U.S.A. Rabbit anti-human y-IFN antibody and y-IFN were kindly supplied by Dr. M. De Ley, Rega Institute, Belgium.
Autologous mixed lymphocyte reaction (AMLR) MNC (2 × 10S/well) as responders and mitomycin C (40 /ag/ml)-treated MNC (2 × 10S/well) as stimulators were incubated in the presence or absence of CCA in flat-bottomed microplates at 37°C for 7 days. In some experiments, appropriate amounts of interleukin 1 and 2 were added to the cultures. Cellular proliferation was determined by 3H-thymidine uptake (0.25 KBq/well; specific activity, 185 GBq; New England Nuclear) during the last 6 h of the culture period. The cells were harvested with a semi-automated cell harvester and radioactivity was counted in a liquid scintillation spectrometer.
IL 1 production Plastic adherent cells were cultured in RPMI 1640 with 1% FCS in the presence of LPS (50tag/ml) a n d / or CCA (50 tag/ml) at 37°C for 24 h in a CO2 incubator. The culture fluid was obtained by centrifugation (500 g at 4°C for 25 min).
IL 1 assay Thymocytes from C3H/HeJ mice were used as the target cells. The cells in RPMI 1640 with 5% FCS were incubated in 96-well microculture plates for 72 h at 37°C in a 5% CO2 incubator. IL 1 containing samples were tested in triplicate by the addition of 50 /al of sample per thymocyte culture (Sugawara, Ishizaka & M611er, 1982).
IL 2 production IL 2 containing supernatants were obtained by incubating MNC (2 × 106/ml) in plastic tubes (Falcon plastics; 2058) with Con A (2/ag/ml) and/or CCA (50 tag/ml) for 24 h.
IL 2 assay The T cell growth stimulatory activity of IL 2 was assayed, using IL 2 dependent T cells derived from a PHA-initiated T cell line (Palacios, Sugawara &
Fernandez, 1982b). The cultures containing 50 tal of IL 2 containing supernatants received 1 x 20" viable IL 2 dependent T cells. Cell proliferation was determined by 3H-thymidine uptake during the last 6 h of a 2 day culture period performed at 37°C.
Lymphocyte blastogenic assay MNC (5 x 10S/well) in 200/al of RPMI 1640 with 5°70 FCS were incubated in the presence of PWM (50 ~g/ml) or SAC (10-Sv/v) at 37°C for 4 days in a CO2 incubator. Tritiated thymidine was introduced 6 h before the termination of the culture. Radioactivity was counted in a liquid scintillation spectrometer.
Plaque forming cell (PFC) assay MNC (5 × 10S/well) were cultured in the presence of PWM (50/ag/ml) with or without CCA (20/ag/ml or 50 /ag/ml) for 7 days. PFC was evaluated by means of protein A-SRBC hemolytic plaque assay (Gronowizc, Coutinho & Melchers, 1976). Briefly, 25/A of the cultured cell suspension (5 × l&/ml) was added to 0.2 ml of 0.5% liquid agar with 3% DEAE dextran containing 25 /al of a protein A-coupled SRBC (dilution: 1: 8). Rabbit anti-mouse IgM or IgG (25/A, diluted 1/5) were added to cells in tubes. The above mixture was placed on petri dishes, covered with cover glasses and incubated at 37°C for 4 h. The results were expressed as PFC per 10s viable cells. All the experiments were done in duplicate.
Effect o f CCA on y-interferon production Human y-interferon production was evaluated by PFC assay with some modifications (Gronowicz et al., 1976; Palacios, Martinez-Maza & De Ley, 1983; Sugawara et al., 1984). Rabbit antihuman yinterferon antibody (final dilution: 1:120) was used instead of rabbit antihuman IgM antibody. The results were expressed as y-IFN PFC/106 viable cells. All the assays were conducted using a final dilution of anti human y-IFN antibody of 1:120, since we found this dilution to be adequate for the development of y-IFN PFC.
Statistical analysis Statistical analyses were made using Student's t-test.
RESULTS
Kinetic study o f the inhibitory effect o f CCA on AMLR One-way stimulated AMLR was performed in the presence or absence of various doses of CCA at 37°C
325
I m m u n o p h a r m a c o l o g i c a l study of C C A Table 1. Kinetic study of dose-dependent effect of C C A on AMLR*
Table 3. Effect of C C A on IL 1 production by LPSstimulated h u m a n monocytes*
Addition of C C A
Supernatant source* (dilution: 1:4)
-
Mean ± S.D. (counts/min) 29443 29507 33663 32330 28850 29113 36388 28191 5684 172
-
20 n g / m l 50 n g / m l 100 n g / m l 500 n g / m l 1 Mg/ml 10 Mg/ml 20 Mg/ml 40 Mg/ml 50 Mg/ml
± ± ± ± ± ± ± ± ± ±
442t 3061 801 805 522 1091 3395 2335 372* 12t
*Autologous mixed lymphocyte reaction (AMLR) was performed in the presence or absence of various doses of C C A at 37°C for 7 days. Cellular proliferation was determined by 3H-thymidine uptake during the last 6 h of the culture period. The results express m e a n ± S.D., each in quadruplicate. *Significantly different from control (P<0.01). f o r 7 d a y s . A s s h o w n in T a b l e 1, 50 / a g / m l C C A inhibited AMLR s i g n i f i c a n t l y , w h i l e 20 M g / m l t h r o u g h 10 M g / m l C C A d i d n o t i n h i b i t it. C C A o f 50 M g / m l w a s n o t c y t o t o x i c to h u m a n M N C .
Effect o f addition o f human purified IL 1 and~or IL 2 upon A M L R M N C (2 × 105/well) a n d m i t o m y c i n C (40 M g / m l ) t r e a t e d M N C (2 x 1 0 V w e l l ) w e r e c o c u l t u r e d in t h e p r e s e n c e o r a b s e n c e o f C C A (50 M g / m l ) at 3 7 ° C f o r 7 days. At the same time, the culture was s u p p l e m e n t e d w i t h 5 / A o f h u m a n I L 1 a n d / o r 10/al
Con A (1 Mg/ml)
-C C A (50 Mg/ml) LPS (50 Mg/ml) LPS (50 ~g/ml) and C C A (50 Mg/ml) -
-
C C A (50 ~g/ml) LPS LPS and C C A 5 MI of pure h u m a n IL 5 M1 of pure h u m a n IL and C C A (50/ag/ml) 5 MI of pure h u m a n IL 5/al of pure h u m a n IL and C C A (50 Mg/ml)
1 1 1 1
-
Activity (counts/min) 269 ± 7 281 _ 3 1670 ± 14
+ + + + -
321 6841 7998 24210 26703 3106
± 2 ± 64 + 432 ± 2280 _+ 920 ± 140
+
2932 ± 43 46520 ± 4300
+
46446 _+ 6041
* C 3 H / H e J murine thymocytes (106/well) were incubated in the presence or absence of IL 1 or IL 1 containing supernatant at 37°C for 3 days. Cellular proliferation was evaluated by 3H-thymidine uptake during the last 6 h of the culture period. All the data show m e a n ± S.D., each in quadruplicate. *Plastic adherent mononuclear cells were cultured with LPS (50 Mg/ml) in the presence or absence o f C C A (50/ag/ml) at 37C for one day. Thereafter, the culture fluid was collected by centrifugation at 500 g at 4°C for 25 min. The culture supernatants collected were dialyzed against double distilled water for 3 days. *Significantly different from control at P<0.01.
o f I L 2 to see w h e t h e r I L l a n d / o r I L 2 c a n o v e r c o m e t h e i n h i b i t o r y e f f e c t o f C C A o n A M L R . C C A (50 Mg/ml) b l o c k e d AMLR significantly but its
Table 2. Effect of IL 1 a n d / o r IL 2 upon depressed A M L R induced by CCA* AMLR
ILl
IL2
CCA
Activity (counts/min)
+ + + + + + + +
+ + + +
+ + + +
+ + + + -
7725 ± 139* 2119 ± 184* 9034 ± 475t 11315 ± 233t 14000± 205t 12367 ± 786 11005 ± 535 16935 ± 533
*MNC (2 × 10S/well) and mitomycin C (40 ~g/ml)-treated M N C (2 × 10S/well) were incubated in the presence or absence of C C A in flat-bottomed microculture plates at 37°C for 7 days. The cultures were supplemented with IL 1 (5 Ml) a n d / o r IL 2 (10 Ml) and incubated at 37°C for 7 days. Cell proliferation was determined by 3H-thymidine uptake during the last 6 h o f the culture period. Control values for mitomycin C-treated M N C and untreated M N C alone were 1670 _+ l l 0 (counts/min) and 2538 ± 224 (counts/min), respectively. *Statistically significant compared with control group (,o
Table 4. Effect o f C C A on IL 2 production by C o n Astimulated T cells* Supernatant source (dilution: 1:4) -M N C alone C C A (50 ~g/ml) Con A (2 Mg/ml) 20ul of h u m a n pure IL 2 10ul of h u m a n pure IL 2 20ul of h u m a n pure IL2 and C C A (50/~g/ml) C o n A and C C A
Activity (counts/min) 2341 2496 3771 8245 10933 8932
± 56* ± 94* ± 46 _+ 678 t ± 12 ± 241
8845 ± 2 7984 ± 37t
*One milliliter of 2 x l0 s h u m a n M N C were cultured with C o n A (2 Mg/ml) in the presence or absence o f C C A (50/ag/ ml) at 37°C for one day. The supernatants were collected and were dialyzed against double distilled water for 3 days and their IL 2 activity was assessed in a microassay based upon the IL 2-dependent proliferation of a PHA-initiated T cell line (3 x 103/well). *Significantly different from control (P<0.01).
326
MASAO FUJIMOTO et al. Table 5. Effect of C C A on D N A synthetic responses induced by P W M or SAC* M N C (5 x 10S/well)
SAC (10-3v/v)
P W M (50/ag/ml)
+ + -
+ +
1562 33387 8747 2050 29022 11295
+ 53 +_ 3665* + 91' _+ 56 _+ 613' +_ 191"
+ + -
+ +
2283 27819 14313 1980 32831 13000
_+ 114 + 416' + 117" _+ 33 +_ 271' _+ 278*
Experiment 1 + + + + CCA(50 ~g/ml) + CCA(50/ag/ml) + CCA(50/ag/ml) Experiment 2 + + + + CCA(50/ag/ml) + CCA(50/ag/ml) + CCA(50 gg/ml)
Activity (counts/min)
*MNC (5 × 10S/well) were cultured for 4 days with or without SAC, P W M a n d / o r CCA. D N A synthesis was evaluated by 3H-thymidine uptake during the last 6 h of the incubation period. All the values show m e a n _+ S.D., each in quadruplicate. *Not significant compared with control at P<0.01. *Not significant compared with control at P<0.001.
i n h i b i t o r y e f f e c t o n A M L R c o u l d be o v e r c o m e at a d d i t i o n o f h u m a n I L 1 a n d / o r I L 2 ( T a b l e 2).
Effect o f CCA on IL 1 generation C 3 H / H e J m u r i n e t h y m o c y t e s (1 x l & / w e l l ) were i n c u b a t e d in t h e p r e s e n c e o r a b s e n c e o f purified IL 1 or I L 1 c o n t a i n i n g s u p e r n a t a n t s f r o m v a r i o u s sources. C C A ( 5 0 / a g / m l ) a b r o g a t e d g e n e r a t i o n o f IL 1 b y L P S s t i m u l a t e d h u m a n m o n o c y t e s s i g n i f i c a n t l y (Table 3).
P H A - i n i t i a t e d T cell line. CCA d i d n o t b l o c k p r o d u c t i o n o f I L 2 b y h u m a n l y m p h o c y t e s ( T a b l e 4).
Effect o f CCA upon DNA synthesis induced by P W M or S A C We examined whether CCA blocked DNA s y n t h e t i c r e s p o n s e s to P W M , T cell d e p e n d e n t B cell m i t o g e n a n d S A C , T cell i n d e p e n d e n t B cell m i t o g e n . A s d e s c r i b e d in T a b l e 5, C C A (50 # g / m l ) d i d n o t inhibit DNA synthesis by PWM or SAC.
Effect o f CCA on IL 2 production O n e milliliter o f 2 x 10 ~ h u m a n M N C w e r e c u l t u r e d in t h e p r e s e n c e o r a b s e n c e o f C C A ( 5 0 / a g / m l ) at 3 7 ° C f o r 24 h . T h e s u p e r n a t a n t s w e r e c o l l e c t e d a n d t h e i r I L 2 a c t i v i t y w a s a s s e s s e d in a m i c r o a s s a y based upon the IL 2 dependent proliferation of a
Effect o f CCA on P WM-driven B-cell differentiation S i n c e we f o u n d t h a t C C A b l o c k e d I L 1 p r o d u c t i o n and speculated that CCA blocked B-cell d i f f e r e n t i a t i o n , we i n v e s t i g a t e d t h e e f f e c t o f C C A o n
Table 6. Effect of C C A upon immunoglobulin plaque forming cells (PFC)* M N C (3 × 10S/well)
P W M (50 ~g/ml)
CCA
IgM PFC/106
lgG PFC/106
+ + + + + +
+ + + -
20 g g / m l 20gg/ml 50gg/ml 50 ~g/ml
33 731' 680 40 33* 33
66 890* 753 53 53* 45
*MNC (3 × 10S/well) were cultured with or without P W M a n d / o r C C A at 37°C for 7 days. SRBC-protein A plaque assay was utilized to enumerate IgM and lgG PFC. All the experiments were done in duplicate. *Significantly different from control (P<0.01).
Immunopharmacological study of CCA Table 7. Effect of CCA on y-IFN producing cells
327
DISCUSSION
We tried to clarify the mechanism of action of C C A . We speculated that C C A had inhibitory effects on T cells and H L A - D R bearing B cells and monocytes, since C C A inhibited proliferation of T cells in A M L R significantly (Palacios & M611er, 1981). Therefore, we performed experiments aiming at investigating whether IL 1 a n d / o r IL 2 can overcome inhibitory effects o f C C A on A M L R , IL 1 or IL 2 addition could reverse depressed A M L R . Thus, we conjectured that C C A was acting on T cells a n d / o r H L A - D R bearing cells. Next, to determine the site of action of C C A we studied whether C C A blocked IL 1 a n d / o r IL 2 production. C C A did not hinder IL 2 production by T cells, but prevented stimulated m a c r o p h a g e s / monocytes from production of IL 1. H o w do we interpret the data that 50/ag/ml C C A blocked P W M - d r i v e n B cell differentiation? It is likely that P F C responses decreased due to decrease of IL 1 and y-interferon did not affect P F C responses, for 20 /ag/ml CCA, inducing y-IFN production, did not block P F C responses induced by P W M . C C A did not affect B cells, since D N A synthetic responses induced by S A C and P W M were not affected with C C A . Therefore, we conjectured from our data that C C A mainly acted on T cells and monocytes. We reported previously that hydrocortisone abrogated proliferation of T cells in A M L R by rendering the IL 2 producer T cells unresponsive to IL 1 and unable to synthesize IL 2 (Palacios & Sugawara, 1982a). In our present study C C A had two actions--induction of y-IFN production and abrogation of IL 1 production. Thus, the mechanism o f action of C C A is completely different from that o f hydrocortisone. C C A is thought to be a promising chemical as an immunomodulator.
In the reported experiments here we showed that C C A was a good in vitro inducer for y-IFN production and abrogated IL 1 production by stimulated human monocytes.
Acknowledgements -- We gratefully acknowledge Dr. M. De Ley, Rega Institute, Belgium, for providing us with human yinterferon and rabbit antihuman y-interferon antibody.
Cell assay
y-IFN PFC/106 viable cells No.1 No.2 No.3
Experiment 1" MNC 350* 210' 53* MNC + Con A(2 Cg/ml 644* 429* 266* MNC+CCA(20ng/ml) 993* 756* 523* MNC(without C' ) 1 0 0 MNC(without anti y-IFN Ab) 0 0 0 MNC + Con A(without C') 0 0 0 MNC + Con A (without antiy-IFN Ab) 0 0 0 Experiment 2* MNC 912' MNC + Con A(2/ag/ml) 3264* MNC + CCA(20/ag/ml) 3840* *Human mononuclear cells were incubated in RPMI 1640 containing 5°70 FCS with or without various chemicals indicated above for one day. Thereafter, the cells were washed and resuspended in BSS. The number of IFN-yPFC was determined by a reverse hemolytic plaque assay using a purified anti IFN-y antibody (final dilution: l:120) as developing sera. In some experiments, neither C' nor anti IFN-y antibody was added to the plaque assay. *Human mononuclear cells were cultured in culture tubes in the presence or absence of a stimulant for two days at 37°C in a CO2 incubator. *Significantly different from controls (P<0.01). P W M - d r i v e n B cell differentiation in terms of plaque forming cells (PFC). As expected, 20/ag/ml C C A did not abrogate generation of P F C , whereas 5 0 / a g / m l C C A abrogated it (Table 6). Effect o f CCA on y-IFN production We studied whether C C A induced y-interferon production in terms of y-IFN P F C . As shown in Table 7, both 20 n g / m l and 20 tag/ml C C A induced y-interferon significantly, y-IFN production by C C A was comparable with that by Con A.
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