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Dose —Response effects of 4-hydroperoxycyclophosphamide on human T and B cell function in vitro
Int. J. lmmunopharmac., Vol. 7, No. 4, pp. 555-560, 1985. Printed in Great Britain.
0192-0561/85 $3.00+ .00 @ 1985 International Society for Immunopharmacology.
D O S E - R E S P O N S E EFFECTS OF 4 - H Y D R O P E R O X Y C Y C L O P H O S P H A M I D E ON H U M A N T A N D B CELL F U N C T I O N I N VITRO HIDEKI KODO,* BENJAMIN BONAVIDA, MICHAEL COLVIN and ROBERT PETER GALE Departments of Medicine (Hematology and Oncology), Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. and Department of Pharmacy, John Hopkins University School of Medicine, Baltimore, MD 21203, U.S.A. (Received 14 June 1984 and in final form 8 November 1984)
Abstract - - 4-Hydroperoxycyclophosphamide (4-OOH-CYP) is spontaneously converted in aqueous solution to 4-hydroxycyclophosphamide (4-OH-CYP), the major active metabolic of cyclophosphamide. We studied the dose related effects of in vitro treatment with 4-OOH-CYP on human T- and B cell-mediated immune responses. T-cell proliferation to mitogens and alloantigens was only partially inhibited even relatively high-doses of 4-OOH-CYP(>6 - 12 tag/ml). In contrast cytotoxic functions of activated T-cells and natural killer (NK) cells were inhibited at lower doses (3 - 6 #g/ml). PWM induced in vitro synthesis of IgG by B-cells was inhibted at < 3 gg/ml of 4-OOH-CYP. These data indicate that 4-HOO-CYP has selective, dose-dependent effects on human T and B cells in vitro.
Cyclophosphamide is an alkylating agent widely used as an immunosuppresive and anti-tumor drug, (Shand, 1979; Friedman, Myles & Colvin, 1979). Cyclophosphamide is inactive in vitro and requires metabolic conversion for biologic activity. Because of this in vitro correlates of cyclophosphamide induced immune suppression have been difficult to investigate. 4-Hydroperoxycyclophosphamide (4O O H - C Y P ) , a derivative of cyclophosphamide, is spontaneously converted in aqueous solution to 4hydroxycyclophosphamide (4-OH-CYP). The latter is believed to be one of the major metabolites of cyclophosphamide (Colvin, Padgett & Fenselou, 1973; Connors, Cox, Farmer, Foster & Jarman, 1974) in vivo and is reported to act in vitro similar to cyclophosphamide activated by rat liver microsomes (Shand & Howard, 1979; Shand, 1972). The present
report describes the dose-dependent effects of 4O O H - C Y P in vitro on human T- and B cell-mediated in vitro immune responses. EXPERIMENTAL PROCEDURES
Isolation o f cells
Peripheral blood (50 ml) was obtained from healthy volunteers, diluted with two volumes of phosphate-buffered saline (PBS), layered in 10 ml aliquots on a F i c o l l - H y p a q u e density gradient and centrifuged 30 min at 400 g (Boyum, 1968). Peripheral blood mononuclear cells (PBMC) were recovered from the interface, resuspended in complete medium (RPMI-1640 with 15% fetal calf serum and 2 m M glutamine) and washed twice. In some experiments blood samples were incubated
*Present address: Department of Medicine, The Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108, Japan. 555
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H. KODOet al.
with carbonyl iron (10/ag/ml) for 30 min at 37°C prior to F i c o l l - H y p a q u e separation to deplete adherent cells.
4-Hydroperoxycyclophosphamide (4-OOH-C YP) treatment 4-OOH-CYP was synthesized by one of us as previously reported (Colvin et al., 1973). The drug was stored as a desiccated powder at - 7 0 ° C and dissolved in RPMI-1640 immediately prior to use. 5 - 1 0 x 106 PBMC or PBL in RPMI-1640 were incubated with a range of concentrations of 4-OOHCYP for l h at 37°C in a humidified 5% CO2 atmosphere. After treatment, PBMC were washed three times with complete medium (RPMI-1640 + 10% FCS) and adjusted to the appropriate viable cell concentration. Viability was determined by trypanblue dye-exclusion test. No immediate cytotoxicity of 4-OOH-CYP was detected after drug treatment at doses ~< 12/ag/ml.
mictotiter plates (Titrek, 76-213-05, Flow Laboratories, Inglewood, CA). After loading effector and target, cell plates were centrifuged at 200 g for 5 rain, and incubated for 4 h at 37°C. The plates were then centrifuged at 600 g for 10 rain. 100 ~1 of supernatant was collected using a multiple analysis sample harvester and radioactivity determined by gamma counting. Natural killer cell (NK) activity was determined in 4-h s'Cr-release assay using K562 cells as targets (Silva, Bonavida & Targan, 1980). Per cent cytotoxicity was calculated as follows: experimental counts/rain - spontaneous counts/rain × 100. maximum counts/rain spontaneous counts/rain Spontaneous counts/min were consistently < 15% of maximum releasable counts. Results of cytotoxicity assays were expressed as lytic units (LU) per 107 cells where a LU is determined as the number of effector cells required for 30°7o lysis of target cells (Cerottini, Engers, Macdonald & Brunner, 1974).
Responses to mitogens and alloantigens
In vitro synthesis o f IgG
1 x l0 s PBMC were cultured with 2 /al of phytohemagglutinin (PHA; Wellcome Research Laboratories, Beckenham, U.K.), concanavalin A (Con A; Sigma, St. Louis, MO) or pokeweed mitogen (PWM; Gibco, Grand Island, NY) in microplates (Irvine Scientific, Santa Ana, CA) for 4 days at 37°C in 5% CO2 atmosphere. For mixed lymphocyte cultures (MLC), 5 x 104 PBMC were cultured with an equal number of allogeneic PBMC in microplates for 4,6,8, or 10 days at 37°C. Stimulator PBMC were inactivated by radiation (3000 R) using a 6°Co-gamma cell. 1 /~Ci of 3Hthymidine (3H-TdR) was added into each well for the final 18 h of culture. In some experiments, 4-HCY was added on day 4 of MLC the fresh stimulator cells were added, and the cultures continued for 3 additional days.
2 x 106 PBMC were cultured with PWM (1/100) in 1.5 ml of complete medium for 7 days in 37°C in a 5% CO2 atmosphere. Synthesis of IgG was determined by radioimmunoassay previously described (Saxon, Stevens & Ashman, 1977). The results of IgG synthesis was expressed as ng per culture. In some experiments, B ceils were isolated by removal of T-cells using AET treated sheep erythrocytes and subsequent buoyant density centrifugation on Fi co l l - H y p aq u e. B- enriched populations (>85%) were treated with 4-OOH-HCY (3/ag/ml) and readded to autologous untreated T cells at a ratio of 1:4. PWM was then added and IgG synthesis determined as indicated.
Cell-mediated lympholysis (CML) CML of PBMC to allogeneic cells was measured in a 4 h S'Cr-release assay using P H A treated blasts as targets. In the sensitization phase, MLCs were performed as described for 7 days. Target cells were prepared as follows. 5 x 106 PBMC from the same donor as the original stimulator cells were cultured with P H A for 3 days, incubated with 150 uCi of s'Cr for 1 h at 37°C and washed twice with complete medium. Cytoxicity assays were performed at four effector: target ratios using target cells in U-shaped
Statistics Experiments were performed in triplicate or quadruplicate as indicated. Experimental groups were compared by two-tailed chi-square or by the Fisher exact test.
RESULTS
Proliferative response to mitogens PBMC, either untreated or treated with 3 - 12/ag/ ml of 4-OOH-CYP, were incubated with PHA, Con A, or PWM and proliferative response was assayed by 3H-TdR incorporation (Table 1). Proliferative
557
4-Hydroperoxycyclophosphamide response to P H A was unaffected at 4 - O O H - C Y P levels ~< 6/ag/ml. At levels ~< 9 ~ g / m l 3H-TdR incorporation was decreased to approximately 60°7o of control. Proliferative responses to Con A and P W M were more sensitive to inhibition with 4 - O O H CYP; significant inhibition was detected at levels ~< 3 Mg/ml (P < 0.05) but remained at 30-40O7o of controls even at 12/ag/ml 4 - O O H - C Y P . Higher concentrations of 4 - O O H - C Y P were directly cytotoxic to P B M C and could not be evaluated.
treating P B M C prior to and following /'n vitro sensitization to allogeneic cells (Table 4). Treatment with 4 - O O H - C Y P prior to sensitization resulted in significant inhibition of C M L only at concentrations > 6 Mg/ml; lower concentrations were without effect. Essentially similar results were observed with treatment with 4 - O O H - C Y P following sensitization. Cytotoxicity in the NK cell assay was significantly decreased at all concentrations tested (Table 4).
Proliferative response in M L C In the initial experiments, P B M C were treated with 4 - O O H - C Y P prior to coculture with allogeneic irradiated stimulator cells in a modified mixed leukocyte reaction. 3H-TdR incorporation was determined on days 6, 8 and 10 (Table 2). Day 6 M L C reactivity was significantly decreased in a dosedependent manner ( P < 0.01). In contrast, M L C reactivity on day 8 was not significantly affected except at a 4 - O O H - C Y P concentration of 12 tag/ml. M L C reactivity on day 10 was significantly higher than controls at all concentrations tested. In a second series of experiments, P B M C were removed from M L C s on day 4, treated with various concentrations 4 - O O H - C Y P , washed and recultured in the presence or absence of stimulator cells for 3 additional days (Table 3). Under these conditions there was a p r o f o u n d dose dependent inhibition of 3H-TdR incorporation. Doses of as low as 6 Mg/ml of 4 - O O H - C Y P resulted in a > 90°70 decrease in proliferation. Inhibition was unrelated to an effect on the stimulator cells since the magnitude of inhibition was similar whether or not fresh stimulator cells were added following 4 - O O H - C Y P treatment (Table 3).
IgG synthesis In these experiments we evaluated the effect of 4O O H - C Y P treatment on P W M induced in vitro synthesis of IgG by cultures containing T and B cells. Treatment with 4 - O O H - C Y P either had no effect or increased IgG synthesis (data not shown). Since this in vitro system is complex we isolated B cells and treated them directly with 3 / a g / m l of 4 - O O H - C Y P . Untreated T-cells were readded, cultures stimulated with P W M , and Ig synthesis determined. Under these conditions IgG synthesis decreased from 16.5 +_ 2.1 to 4.2 _+ 0.3 ng per culture (P < 0.01).
Cytotoxic activity The effects of 4 - O O H - C Y P on precursor and mature cytotoxic cells in C M L were studied by
DISCUSSION We studied d o s e - r e s p o n s e effects of 4-OOHCYP, an active metablite of cyclophosphamide, on human T and B cells in vitro. Our findings can be summarized as follows: (1) proliferative response to mitogens was only partially inhibited at concentrations 3 - 1 2 / a g / m l ; P H A reactivity was relatively less sensitive to inhibition than were Con A or P W M reactivity; (2) proliferative response to alloantigens on day 6 (as determined in M L C test) was inhibited by concentrations >/ 3 tag/ml; day 8 and day 10 M L C reactivity were unaffected or increased; (3) when alloantigen reactive cells were treated with 4 - O O H - C Y P after day 4 of M L C , they
Table 1. Effects of 4-OOH-CYP on mitogenic responses* Mitogen 4-OOHCYP None
PHA
Con A
PWM
0 3 6 9 12
13,237 --. 1815 (100)# 11,705 ± 2011 (88) 12,114+ 1698 (92) 8338 ± 674 (63) 8362 +- 1335 (63)
17,737 - 4616 (100) 12,840+- 1210 (72) 12,466± 72 (70) 7342 ± 1835 (41) 6494 + 145 (37)
9086 5725 5600 3608 2955
195 397 316 185 276
± 4t _+ 247 --- 76 ± 43 + 1
*4-Hydroperoxycyclophosphamide (~g/ml). tMean _+ S.E.M. #Per cent of control.
_+ 2729 (100) _+ 95 (63) _+ 375 (62) _+ 160 (40) -+ 313 (33)
558
H. KoDo et aL Table 2. Effect of 4-OOH-CYP on mixed lymphocyte culture 4-OOH-CYP
Days of culture 8
6 0 3 6 9 12
20,107 14,615 12,253 9321 7679
+ 1485 (100)* +__130 (73) _.+ 529 (61) _+ 439 (46) + 456 (38)
32,403 24,711 30,104 29,711 21,920
10
__. 1452 (100) +_ 492 (76) + 1274 (93) _+ 822 (92) __+965 (68)
9091 14,992 17,987 12,057 14,800
+ 569 (100) +_ 307 (165) +__248 (198) _+ 21 (133) __. 173 (163)
*Per cent of control. Table 3. Effects of 4-OOH-CYP on activated cells in mixed lymphocyte culture 4-OOH-CYP 0 3 6 9 12
No added stimulators
Added stimulators
33,773 16,746 2635 278 103
71,209 37,789 7629 851 303
_+ 324 (100)* __. 1353 (50) _+ 189 (8) _+ 65 (1) ___5
_+ 1992 (100) ___2638 (53) _+ 230 (11) _+ 73 (1) + 44
*Per cent of control. Table 4. Effects of 4-OOH-CYP on cytotoxic cells 4-OOH-CYP1
0 1
3 6 9 12
Cytoxic activity Treatment Treatment prior to MLC following MLC 40 (100)* ND 39 (98) 31 (78) 20 (50) 18 (45)
22 (100)'t 23 (105) 21 (95) 11 (50) 14 (64) 7 (32)
NK activity
90 (100) ND:I: 40 (44) 20 (22) 11 (12) 4 (4)
*Lytic units. 5Per cent of control. ~ND not determined.
were extremely sensitive to inhibition whether or not fresh s t i m u l a t o r cells were added; (4) precursor cells o f C T L , as well as C T L themselves, were sensitive to inhibition by 4 - O O H - C Y P at c o n c e n t r a t i o n s > 6 - 9 / a g / m l ; only 50°70 inhibition was observed at a dose of 12/ag/ml. NK cells were m o r e sensitive with 50070 inhibition at 3 tag/ml a n d > 90070 inhibition at 12/ag/ml; (5) in vitro IgG synthesis by P W M stimulated B cells was inhibited by low c o n c e n t r a t i o n s (3/~g/ml) o f 4 - O O H - C Y P . O u r findings using 4 - O O H - C Y P are in most instances similar to data obtained using m i c r o s o m a l l y activated c y c l o p h o s p h a m i d e in vitro a n d the p a r e n t c o m p o u n d in vivo ( S h a n d , 1972). T h e
o b s e r v a t i o n that 4 - O O H - C Y P was most active against proliferating cells is typical o f alkylating agents ( F r i e d m a n et al., 1979). Interestingly, we observed a differential sensitivity o f mitogen reactive cells with P H A reactivity being m o r e resistant to inhibition by 4 - O O H - C Y P when c o m p a r e d to C o n A a n d P W M . Ozer et al., r e p o r t e d d a t a o n mitogen induced proliferation following t r e a t m e n t with 4 - O O H - C Y P (Ozer, Cowens, Colvin, N u s s b a u m B l u e m s o n & S h e e d y , 1982). Parallel studies have also been r e p o r t e d in mice using lipopolysaccharide (Diamantstein, Willinger & Reiman, 1979). A l t h o u g h there are no reports regarding the effect of 4 - O O H - C Y P on NK cells in vitro, c y c l o p h o s p h a m i d e
4-Hydroperoxycyclophosphamide is a potent inhibitor of NK activity in vivo (Lust, Rumar, Burton, Bartlett & Bennett, 1981). It has also been reported that the precursors o f NK cells are insensitive to cyclophosphamide but that active NK cells are sensitive (Dieu, Heinbaugh, Vieira, Holden & Herberman, 1979). Our data are in agreement in that NK cells were inhibited by treatment with 4 - O O H - C Y P at low doses. We found a disparity in the effect of 4 - O O H - C Y P on P W M - i n d u c e d IgG synthesis when unfractionated P B M C were treated compared to B-cell enriched populations. Treatment of the former had no effect on IgG levels whereas treatment of B-cells markedly decreased IgG synthesis. A parallel observation has been made by Ozer and coworkers (1982) who reported that 4 - O O H - C Y P induced inhibition of B-cells can be reversed by readdition of 4 - O O H - C Y P treated T-cells. We interpret these data to indicate that in mixed cell populations the predominant effect of 4 - O O H - C Y P treatment is to inhibit the development of T-suppressor cells with a resultant increase in P W M stimulated Ig synthesis. When B-cells are treated alone they are inhibited and this is not compensated for by inhibition of Tsuppressor cells and the net effect is inhibition of lg
559
synthesis. Detailed studies o f the effects of 4-OOHCYP on murine T cytotoxic cell progenitors in vitro support this concept (Cowens, Ozer, Ehrke, Greco, Colvin & Mihich, 1984). In conclusion, we found that 4 - O O H - C Y P inhibits various functions o f human T and B cells functions including mitogen and alloantigen induced T-cell proliferation, cytotoxic and NK cell function, and PWM-induced IgG synthesis by isolated B-cells. Inhibition of these functions occurred at different dose of 4 - O O H - C Y P . The exact mechanisms involved as well as the reversibility of the immune suppression are areas currently being investigated. The findings that there are selective immune suppressive effect dependent on the dose of 4 - O O H CYP used suggests it may be possible to vary the dose of cyclophosphamide used in vivo to produce differential immune suppression. Acknowledgement - - This study was supported in part by
Grants CA 23175 from the NCI, NIH, USPHS, DHSS and in part by the Concern Foundation at Los Angeles. Dr. Ross Abrams kindly reviewed the manuscript and it was typed by Celene Ushry and Shaun Taylor. Dr. Andrew Saxon helped with the Ig assay.
REFERENCES
BOYUM,A. (1968). Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. clin. Lab, Invest., Suppl. 97, 77-89.
CEROTTZN1,J. C., ENTERS,H. D., MACDONALD,H. R. & BRUNNER,K. T. (1974). Generation of cytotoxic T lymphocytes in vitro. I. Response of normal and immune mouse spleen cells in mixed leukocyte cultures. J. exp. Med., 140, 703 - 717. COLVIN,M., PADGETT,C. m. & FENSELOU,C. (1973). A biologically active metabolite of cyclophosphamide. Cancer Res., 33, 915. CONNORS,T. A., COX, P. J., FARMER,F. B., FOSTER,A. B. & JARMAN,M. (1974). Some studies of the active intermediates formed in the microsomal metabolism of cyclophospharnide and and isophosphamide. Biochem. Pharmac., 23, 115. Pharmac., 23, 115. COWENS, J. W., OZER, H., EHRKE, M. J., GRECO, W. R., COLVlN,M. & MIHtCH, E. (1984). Inhibition of the development of suppressor cells in culture by 4-hydroperoxycyclophosphamide. J. lmmun., 132, 95-100. DIAMANTSTEIN,T., WILLINGER,E. & REIMAN,J. (1979). T-suppressor cells sensitive to cyclophosphamide and to its in vitro active derivative 4-hydroperoxycyclophosphamide control the mitogenic response of murine splenic B cells to dextran sulphate: A direct proof for different sensitivities of lymphocyte subsets to cyclophosphamide. J. exp. Med., 150, 1571 - 1576. DJEU, J. Y., HEINBAUGH, J. A., VIEIRA, W. D., HOLDEN, H. T. & HERBERMAN, R. B. (1979). The effect of immunopharmacological agents on mouse natural cell-mediated cytotoxicity and on its augmentation by poly I'C. lmmunopharmacology, 1, 231-244. FRIEDMAN, D. M., MYLES, A. & COLVlN, M. (1979). Cyclophosphamide and related phosphoramide mustards: Current status and future prospects. In Advances in Cancer Chemotherapy (ed. Rosowsky, A.) pp. 143-203, Marcel Dekker, New York. LUST, J. A,, RUMAR, V., BURTON,R. C. M., BARTLETT,S. D. & BENNETT,M. (1981). Heterogeneity of natural killer cells in the mouse. J. exp. Med., 154, 306-317. OZER, H., COWENS, J. W., COLVIN, M., NUSSBAUM-BLUEMSON,A. & SbIEEDY, D. (1982). In vitro effects 4hydroperoxycyciophosphamide on human immunoregulatory T subset function. I. Selective effects on lymphoctye function in T-B cell corporation. J. exp. Med., 155, 276-290.
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SAXON, A., STEVENS,R. H. & ASHMAN,R. F. (1977). Regulation of immunoglobulin production in human peripheral blood leukocytes: Cellular interactions. J. I m m u n . , 118, 1872- 1879. SHA~D, F. L. (1972). The capacity of microsomally-activated cyclophosphamide to induce immunosuppression in vitro. Immunology, 35, 1017-1025. SHA~qD, F. L. (1979). The immunopharmacology of cyclophosphamide. Int. J. Immunopharmac., l, 165-171. SHAND, F. L. • HOWARD, J. G. (1979). Induction in vitro of reversible immunosuppression and inhibition of B cell receptor regeneration by defined metabolites of cyclophosphamide. Eur. J. l m m u n . , 9, 17-21. SILVA, D., BONAVIDA, B. & TARGAN, S. (1980). Mode of action of interferon-mediated modulation of natural killer cytotoxic activity: Recruitment of pre-NK cells and enhanced kinetics of lysis. J. I m m u n . , 125, 479-484.
0192-0561/85 $3.00+ .00 @ 1985 International Society for Immunopharmacology.
D O S E - R E S P O N S E EFFECTS OF 4 - H Y D R O P E R O X Y C Y C L O P H O S P H A M I D E ON H U M A N T A N D B CELL F U N C T I O N I N VITRO HIDEKI KODO,* BENJAMIN BONAVIDA, MICHAEL COLVIN and ROBERT PETER GALE Departments of Medicine (Hematology and Oncology), Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. and Department of Pharmacy, John Hopkins University School of Medicine, Baltimore, MD 21203, U.S.A. (Received 14 June 1984 and in final form 8 November 1984)
Abstract - - 4-Hydroperoxycyclophosphamide (4-OOH-CYP) is spontaneously converted in aqueous solution to 4-hydroxycyclophosphamide (4-OH-CYP), the major active metabolic of cyclophosphamide. We studied the dose related effects of in vitro treatment with 4-OOH-CYP on human T- and B cell-mediated immune responses. T-cell proliferation to mitogens and alloantigens was only partially inhibited even relatively high-doses of 4-OOH-CYP(>6 - 12 tag/ml). In contrast cytotoxic functions of activated T-cells and natural killer (NK) cells were inhibited at lower doses (3 - 6 #g/ml). PWM induced in vitro synthesis of IgG by B-cells was inhibted at < 3 gg/ml of 4-OOH-CYP. These data indicate that 4-HOO-CYP has selective, dose-dependent effects on human T and B cells in vitro.
Cyclophosphamide is an alkylating agent widely used as an immunosuppresive and anti-tumor drug, (Shand, 1979; Friedman, Myles & Colvin, 1979). Cyclophosphamide is inactive in vitro and requires metabolic conversion for biologic activity. Because of this in vitro correlates of cyclophosphamide induced immune suppression have been difficult to investigate. 4-Hydroperoxycyclophosphamide (4O O H - C Y P ) , a derivative of cyclophosphamide, is spontaneously converted in aqueous solution to 4hydroxycyclophosphamide (4-OH-CYP). The latter is believed to be one of the major metabolites of cyclophosphamide (Colvin, Padgett & Fenselou, 1973; Connors, Cox, Farmer, Foster & Jarman, 1974) in vivo and is reported to act in vitro similar to cyclophosphamide activated by rat liver microsomes (Shand & Howard, 1979; Shand, 1972). The present
report describes the dose-dependent effects of 4O O H - C Y P in vitro on human T- and B cell-mediated in vitro immune responses. EXPERIMENTAL PROCEDURES
Isolation o f cells
Peripheral blood (50 ml) was obtained from healthy volunteers, diluted with two volumes of phosphate-buffered saline (PBS), layered in 10 ml aliquots on a F i c o l l - H y p a q u e density gradient and centrifuged 30 min at 400 g (Boyum, 1968). Peripheral blood mononuclear cells (PBMC) were recovered from the interface, resuspended in complete medium (RPMI-1640 with 15% fetal calf serum and 2 m M glutamine) and washed twice. In some experiments blood samples were incubated
*Present address: Department of Medicine, The Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108, Japan. 555
556
H. KODOet al.
with carbonyl iron (10/ag/ml) for 30 min at 37°C prior to F i c o l l - H y p a q u e separation to deplete adherent cells.
4-Hydroperoxycyclophosphamide (4-OOH-C YP) treatment 4-OOH-CYP was synthesized by one of us as previously reported (Colvin et al., 1973). The drug was stored as a desiccated powder at - 7 0 ° C and dissolved in RPMI-1640 immediately prior to use. 5 - 1 0 x 106 PBMC or PBL in RPMI-1640 were incubated with a range of concentrations of 4-OOHCYP for l h at 37°C in a humidified 5% CO2 atmosphere. After treatment, PBMC were washed three times with complete medium (RPMI-1640 + 10% FCS) and adjusted to the appropriate viable cell concentration. Viability was determined by trypanblue dye-exclusion test. No immediate cytotoxicity of 4-OOH-CYP was detected after drug treatment at doses ~< 12/ag/ml.
mictotiter plates (Titrek, 76-213-05, Flow Laboratories, Inglewood, CA). After loading effector and target, cell plates were centrifuged at 200 g for 5 rain, and incubated for 4 h at 37°C. The plates were then centrifuged at 600 g for 10 rain. 100 ~1 of supernatant was collected using a multiple analysis sample harvester and radioactivity determined by gamma counting. Natural killer cell (NK) activity was determined in 4-h s'Cr-release assay using K562 cells as targets (Silva, Bonavida & Targan, 1980). Per cent cytotoxicity was calculated as follows: experimental counts/rain - spontaneous counts/rain × 100. maximum counts/rain spontaneous counts/rain Spontaneous counts/min were consistently < 15% of maximum releasable counts. Results of cytotoxicity assays were expressed as lytic units (LU) per 107 cells where a LU is determined as the number of effector cells required for 30°7o lysis of target cells (Cerottini, Engers, Macdonald & Brunner, 1974).
Responses to mitogens and alloantigens
In vitro synthesis o f IgG
1 x l0 s PBMC were cultured with 2 /al of phytohemagglutinin (PHA; Wellcome Research Laboratories, Beckenham, U.K.), concanavalin A (Con A; Sigma, St. Louis, MO) or pokeweed mitogen (PWM; Gibco, Grand Island, NY) in microplates (Irvine Scientific, Santa Ana, CA) for 4 days at 37°C in 5% CO2 atmosphere. For mixed lymphocyte cultures (MLC), 5 x 104 PBMC were cultured with an equal number of allogeneic PBMC in microplates for 4,6,8, or 10 days at 37°C. Stimulator PBMC were inactivated by radiation (3000 R) using a 6°Co-gamma cell. 1 /~Ci of 3Hthymidine (3H-TdR) was added into each well for the final 18 h of culture. In some experiments, 4-HCY was added on day 4 of MLC the fresh stimulator cells were added, and the cultures continued for 3 additional days.
2 x 106 PBMC were cultured with PWM (1/100) in 1.5 ml of complete medium for 7 days in 37°C in a 5% CO2 atmosphere. Synthesis of IgG was determined by radioimmunoassay previously described (Saxon, Stevens & Ashman, 1977). The results of IgG synthesis was expressed as ng per culture. In some experiments, B ceils were isolated by removal of T-cells using AET treated sheep erythrocytes and subsequent buoyant density centrifugation on Fi co l l - H y p aq u e. B- enriched populations (>85%) were treated with 4-OOH-HCY (3/ag/ml) and readded to autologous untreated T cells at a ratio of 1:4. PWM was then added and IgG synthesis determined as indicated.
Cell-mediated lympholysis (CML) CML of PBMC to allogeneic cells was measured in a 4 h S'Cr-release assay using P H A treated blasts as targets. In the sensitization phase, MLCs were performed as described for 7 days. Target cells were prepared as follows. 5 x 106 PBMC from the same donor as the original stimulator cells were cultured with P H A for 3 days, incubated with 150 uCi of s'Cr for 1 h at 37°C and washed twice with complete medium. Cytoxicity assays were performed at four effector: target ratios using target cells in U-shaped
Statistics Experiments were performed in triplicate or quadruplicate as indicated. Experimental groups were compared by two-tailed chi-square or by the Fisher exact test.
RESULTS
Proliferative response to mitogens PBMC, either untreated or treated with 3 - 12/ag/ ml of 4-OOH-CYP, were incubated with PHA, Con A, or PWM and proliferative response was assayed by 3H-TdR incorporation (Table 1). Proliferative
557
4-Hydroperoxycyclophosphamide response to P H A was unaffected at 4 - O O H - C Y P levels ~< 6/ag/ml. At levels ~< 9 ~ g / m l 3H-TdR incorporation was decreased to approximately 60°7o of control. Proliferative responses to Con A and P W M were more sensitive to inhibition with 4 - O O H CYP; significant inhibition was detected at levels ~< 3 Mg/ml (P < 0.05) but remained at 30-40O7o of controls even at 12/ag/ml 4 - O O H - C Y P . Higher concentrations of 4 - O O H - C Y P were directly cytotoxic to P B M C and could not be evaluated.
treating P B M C prior to and following /'n vitro sensitization to allogeneic cells (Table 4). Treatment with 4 - O O H - C Y P prior to sensitization resulted in significant inhibition of C M L only at concentrations > 6 Mg/ml; lower concentrations were without effect. Essentially similar results were observed with treatment with 4 - O O H - C Y P following sensitization. Cytotoxicity in the NK cell assay was significantly decreased at all concentrations tested (Table 4).
Proliferative response in M L C In the initial experiments, P B M C were treated with 4 - O O H - C Y P prior to coculture with allogeneic irradiated stimulator cells in a modified mixed leukocyte reaction. 3H-TdR incorporation was determined on days 6, 8 and 10 (Table 2). Day 6 M L C reactivity was significantly decreased in a dosedependent manner ( P < 0.01). In contrast, M L C reactivity on day 8 was not significantly affected except at a 4 - O O H - C Y P concentration of 12 tag/ml. M L C reactivity on day 10 was significantly higher than controls at all concentrations tested. In a second series of experiments, P B M C were removed from M L C s on day 4, treated with various concentrations 4 - O O H - C Y P , washed and recultured in the presence or absence of stimulator cells for 3 additional days (Table 3). Under these conditions there was a p r o f o u n d dose dependent inhibition of 3H-TdR incorporation. Doses of as low as 6 Mg/ml of 4 - O O H - C Y P resulted in a > 90°70 decrease in proliferation. Inhibition was unrelated to an effect on the stimulator cells since the magnitude of inhibition was similar whether or not fresh stimulator cells were added following 4 - O O H - C Y P treatment (Table 3).
IgG synthesis In these experiments we evaluated the effect of 4O O H - C Y P treatment on P W M induced in vitro synthesis of IgG by cultures containing T and B cells. Treatment with 4 - O O H - C Y P either had no effect or increased IgG synthesis (data not shown). Since this in vitro system is complex we isolated B cells and treated them directly with 3 / a g / m l of 4 - O O H - C Y P . Untreated T-cells were readded, cultures stimulated with P W M , and Ig synthesis determined. Under these conditions IgG synthesis decreased from 16.5 +_ 2.1 to 4.2 _+ 0.3 ng per culture (P < 0.01).
Cytotoxic activity The effects of 4 - O O H - C Y P on precursor and mature cytotoxic cells in C M L were studied by
DISCUSSION We studied d o s e - r e s p o n s e effects of 4-OOHCYP, an active metablite of cyclophosphamide, on human T and B cells in vitro. Our findings can be summarized as follows: (1) proliferative response to mitogens was only partially inhibited at concentrations 3 - 1 2 / a g / m l ; P H A reactivity was relatively less sensitive to inhibition than were Con A or P W M reactivity; (2) proliferative response to alloantigens on day 6 (as determined in M L C test) was inhibited by concentrations >/ 3 tag/ml; day 8 and day 10 M L C reactivity were unaffected or increased; (3) when alloantigen reactive cells were treated with 4 - O O H - C Y P after day 4 of M L C , they
Table 1. Effects of 4-OOH-CYP on mitogenic responses* Mitogen 4-OOHCYP None
PHA
Con A
PWM
0 3 6 9 12
13,237 --. 1815 (100)# 11,705 ± 2011 (88) 12,114+ 1698 (92) 8338 ± 674 (63) 8362 +- 1335 (63)
17,737 - 4616 (100) 12,840+- 1210 (72) 12,466± 72 (70) 7342 ± 1835 (41) 6494 + 145 (37)
9086 5725 5600 3608 2955
195 397 316 185 276
± 4t _+ 247 --- 76 ± 43 + 1
*4-Hydroperoxycyclophosphamide (~g/ml). tMean _+ S.E.M. #Per cent of control.
_+ 2729 (100) _+ 95 (63) _+ 375 (62) _+ 160 (40) -+ 313 (33)
558
H. KoDo et aL Table 2. Effect of 4-OOH-CYP on mixed lymphocyte culture 4-OOH-CYP
Days of culture 8
6 0 3 6 9 12
20,107 14,615 12,253 9321 7679
+ 1485 (100)* +__130 (73) _.+ 529 (61) _+ 439 (46) + 456 (38)
32,403 24,711 30,104 29,711 21,920
10
__. 1452 (100) +_ 492 (76) + 1274 (93) _+ 822 (92) __+965 (68)
9091 14,992 17,987 12,057 14,800
+ 569 (100) +_ 307 (165) +__248 (198) _+ 21 (133) __. 173 (163)
*Per cent of control. Table 3. Effects of 4-OOH-CYP on activated cells in mixed lymphocyte culture 4-OOH-CYP 0 3 6 9 12
No added stimulators
Added stimulators
33,773 16,746 2635 278 103
71,209 37,789 7629 851 303
_+ 324 (100)* __. 1353 (50) _+ 189 (8) _+ 65 (1) ___5
_+ 1992 (100) ___2638 (53) _+ 230 (11) _+ 73 (1) + 44
*Per cent of control. Table 4. Effects of 4-OOH-CYP on cytotoxic cells 4-OOH-CYP1
0 1
3 6 9 12
Cytoxic activity Treatment Treatment prior to MLC following MLC 40 (100)* ND 39 (98) 31 (78) 20 (50) 18 (45)
22 (100)'t 23 (105) 21 (95) 11 (50) 14 (64) 7 (32)
NK activity
90 (100) ND:I: 40 (44) 20 (22) 11 (12) 4 (4)
*Lytic units. 5Per cent of control. ~ND not determined.
were extremely sensitive to inhibition whether or not fresh s t i m u l a t o r cells were added; (4) precursor cells o f C T L , as well as C T L themselves, were sensitive to inhibition by 4 - O O H - C Y P at c o n c e n t r a t i o n s > 6 - 9 / a g / m l ; only 50°70 inhibition was observed at a dose of 12/ag/ml. NK cells were m o r e sensitive with 50070 inhibition at 3 tag/ml a n d > 90070 inhibition at 12/ag/ml; (5) in vitro IgG synthesis by P W M stimulated B cells was inhibited by low c o n c e n t r a t i o n s (3/~g/ml) o f 4 - O O H - C Y P . O u r findings using 4 - O O H - C Y P are in most instances similar to data obtained using m i c r o s o m a l l y activated c y c l o p h o s p h a m i d e in vitro a n d the p a r e n t c o m p o u n d in vivo ( S h a n d , 1972). T h e
o b s e r v a t i o n that 4 - O O H - C Y P was most active against proliferating cells is typical o f alkylating agents ( F r i e d m a n et al., 1979). Interestingly, we observed a differential sensitivity o f mitogen reactive cells with P H A reactivity being m o r e resistant to inhibition by 4 - O O H - C Y P when c o m p a r e d to C o n A a n d P W M . Ozer et al., r e p o r t e d d a t a o n mitogen induced proliferation following t r e a t m e n t with 4 - O O H - C Y P (Ozer, Cowens, Colvin, N u s s b a u m B l u e m s o n & S h e e d y , 1982). Parallel studies have also been r e p o r t e d in mice using lipopolysaccharide (Diamantstein, Willinger & Reiman, 1979). A l t h o u g h there are no reports regarding the effect of 4 - O O H - C Y P on NK cells in vitro, c y c l o p h o s p h a m i d e
4-Hydroperoxycyclophosphamide is a potent inhibitor of NK activity in vivo (Lust, Rumar, Burton, Bartlett & Bennett, 1981). It has also been reported that the precursors o f NK cells are insensitive to cyclophosphamide but that active NK cells are sensitive (Dieu, Heinbaugh, Vieira, Holden & Herberman, 1979). Our data are in agreement in that NK cells were inhibited by treatment with 4 - O O H - C Y P at low doses. We found a disparity in the effect of 4 - O O H - C Y P on P W M - i n d u c e d IgG synthesis when unfractionated P B M C were treated compared to B-cell enriched populations. Treatment of the former had no effect on IgG levels whereas treatment of B-cells markedly decreased IgG synthesis. A parallel observation has been made by Ozer and coworkers (1982) who reported that 4 - O O H - C Y P induced inhibition of B-cells can be reversed by readdition of 4 - O O H - C Y P treated T-cells. We interpret these data to indicate that in mixed cell populations the predominant effect of 4 - O O H - C Y P treatment is to inhibit the development of T-suppressor cells with a resultant increase in P W M stimulated Ig synthesis. When B-cells are treated alone they are inhibited and this is not compensated for by inhibition of Tsuppressor cells and the net effect is inhibition of lg
559
synthesis. Detailed studies o f the effects of 4-OOHCYP on murine T cytotoxic cell progenitors in vitro support this concept (Cowens, Ozer, Ehrke, Greco, Colvin & Mihich, 1984). In conclusion, we found that 4 - O O H - C Y P inhibits various functions o f human T and B cells functions including mitogen and alloantigen induced T-cell proliferation, cytotoxic and NK cell function, and PWM-induced IgG synthesis by isolated B-cells. Inhibition of these functions occurred at different dose of 4 - O O H - C Y P . The exact mechanisms involved as well as the reversibility of the immune suppression are areas currently being investigated. The findings that there are selective immune suppressive effect dependent on the dose of 4 - O O H CYP used suggests it may be possible to vary the dose of cyclophosphamide used in vivo to produce differential immune suppression. Acknowledgement - - This study was supported in part by
Grants CA 23175 from the NCI, NIH, USPHS, DHSS and in part by the Concern Foundation at Los Angeles. Dr. Ross Abrams kindly reviewed the manuscript and it was typed by Celene Ushry and Shaun Taylor. Dr. Andrew Saxon helped with the Ig assay.
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