Suppression of lymphocyte proliferation by hexamethylene diamine

Toxicology, 56 (1989) 301--313 Elsevier Scientific Publishers Ireland Ltd.

SUPPRESSION OF LYMPHOCYTE HEXAMETHYLENE DIAMINE*

PROLIFERATION

BY

ROBERT W. LUEBKE, CAREY B. COPELAND, ORLANDO IRSULA, MARIE M. RIDDLE, RON R. ROGERS, CHRISTOPHER LAU and RALPH J. SMIALOWICZ Perinatal Toxicology Branch, Health Effects Research Laboratory, US E.P.A., Research Triangle Park~ NC 27711 and Northrop Services, Inc., Research Triangle Park~ NC, 27709 fU.S.AJ

(Received August 9th, 1988) (Accepted November 9th, 1988)

SUMMARY T h e a n t i p r o l i f e r a t i v e p o t e n t i a l of h e x a m e t h y l e n e diamine (HMDA) for m i t o g e n - s t i m u l a t e d splenic l y m p h o c y t e s was e v a l u a t e d in v i t r o at final c o n c e n t r a t i o n s of 0.1--16 mM. Addition at the s t a r t of culture or a f t e r 24 or 48 h of c u l t u r e d e c r e a s e d t h e p r o l i f e r a t i v e r e s p o n s e to T and B cell m i t o g e n s . H o w e v e r , t h e c o n c e n t r a t i o n of H M D A r e q u i r e d to cause s u p p r e s sion i n c r e a s e d with incubation time. R e m o v a l of diamine a f t e r 24 h allowed cells to p r o l i f e r a t e n o r m a l l y upon r e c u l t u r e with mitogen. Mitogenic responses of c u l t u r e s containing t h e p o t e n t o r n i t h i n e d e c a r b o x y l a s e (ODC) inhibitor a - d i f l u o r o m e t h y l o r n i t h i n e (DFMO) w e r e also inhibited in a t i m e and dose d e p e n d e n t fashion. ODC a c t i v i t y , which w a s m u c h g r e a t e r in c u l t u r e s s t i m u l a t e d with Con A t h a n L P S , w a s m a r k e d l y d e c r e a s e d b y inclusion of diamine or D F M O in t h e c u l t u r e m e d i u m . Addition of p u t r e s c i n e to c u l t u r e s did not r e v e r s e t h e s u p p r e s s i v e e f f e c t s of diamine on proliferation b u t did r e s t o r e D F M 0 - c o n t a i n i n g c u l t u r e s to control levels of activity. T h e s e r e s u l t s indicate t h a t H M D A does s u p p r e s s l y m p h o c y t e proliferation in v i t r o b y a l t e r a t i o n of ODC and p o l y a m i n e a c t i v i t y . H o w e v e r , c o m p a r i s o n of r e s u l t s o b t a i n e d with D F M O and H M D A s u g g e s t s t h a t H M D A m a y act via multiple p a t h w a y s , only one of which involves inhibition of ODC activity.

*Disclaimer: This paper has been reviewed by the Health Effects Research Laboratory, U.S.

Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. 0300-483x/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

301

Key words: 1,6-Hexamethylene diamine; Ornithine decarboxylase; Polyamine synthesis; DL-a-Difluoromethylornithine; Lymphocyte proliferation-suppression; Putrescine INTRODUCTION 1,6-Hexamethylene diamine (HMDA) is used primarily as starting material in the production of Nylon 66. In the free amine form, HMDA is caustic and classified as a skin irritant. Hematologic and phagocytic defects have been described in rodents following long-term inhalation exposure to HMDA [1] and splenic atrophy and reduced antibody responses were reported in rats exposed to HMDA in drinking water for 1 year [2]. HMDA is a known inhibitor of ornithine decarboxylase (ODC) [3,4] the rate-limiting enzyme in polyamine synthesis. ODC has a central role in cell division and differentiation since polyamine synthesis precedes DNA synthesis (reviewed in [5]) and may regulate the synthesis of RNA by activating RNA polymerase I [6]. Intracellular levels of ODC increase rapidly in lymphocytes following mitogenic stimulation [7,8] but not after exposure to non-mitogenic lectins [8]. The goal of this study was to examine the effects of HMDA on mitogeninduced lymphocyte proliferative responses and stimulation of ODC activity, since in vitro inhibition of ODC activity by HMDA has been described in cell lines [9,10]. To determine whether any effects of HMDA are exerted through an ODC-mediated mechanism, DL-a-difluoromethylornithine (DFMO), a catalytic, irreversible inhibitor of ODC [11], was examined and compared with HMDA. MATERIALSAND METHODS Animals Female C57BL/6J mice were purchased from Jackson Labs (Bar Harbor, ME) at 9 weeks of age. At least 2 weeks were allowed for recovery from shipping stress before use in experiments. Mice were housed on wood chips in plastic cages, maintained at 22 _+ 0.5°C at 50% relative humidity and provided free access to food and water. Chemicals HMDA and its dihydrochloride salt (1,6-hexamethylene diamine dihydrochloride, HDDH) were purchased from Aldrich Chemical Co. (Milwaukee, WS). DFMO was generously provided by Dr. Peter McCann, Merrell Dow Research Institute (Cincinnati, OH). Stock solutions were prepared in RPMI 1640 containing 2 mM L-Glutamine, 25 mM HEPES buffer and 1 ~g/ml gentimycin (GIBCO). Solutions were adjusted to a pH of 7.0 and filter sterilized (0.22 ~m pore size, Nalgene, Rochester, NY) prior to use. Effects on cell proliferation The effects of HMDA, HDDH or DFMO on the proliferative response of 302

lymphocytes was assessed in cultures containing 2 ~g/ml of the T cell mitogens concanavalin A (Con A, Difco Labs, Detroit, MI) or purified phytohemagglutinin (PHA, Burroughs Wellcome, Research Triangle Park, NC) or 20 ~g/ml of the B cell mitogen E. coli lipopolysaccharide (LPS, Difco). Final concentrations of HMDA and HDDH up to 16 mM and DFMO up to 1.0 mM are reported here. Greater concentrations (up to 100 mM) of the 3 compounds were found in pilot studies to be cytotoxic and are therefore not reported. Single cell suspensions of erythrocyte-depleted (NH4C1 lysis) splenocytes were prepared in supplemented RPMI containing 5% human AB serum (Irvine Scientific, Santa Ana, CA) and cultured in a final volume of 220 ~l in flat bottom, 96-well culture plates (Costar, Cambridge, MA). Cell viability was assessed by trypan blue dye exclusion and cell numbers were determined electronically (Coulter model ZB1, Coulter Electronics, Hialeah, FL). Twenty milliliters of the appropriate concentration of chemical solution was added either at the start of culture (0 h) or at the indicated time thereafter (see figure legends} to 2 x 105 viable cells/well. Approximately 68 h after culture initiation, 0.5 ~Ci/well of tritiated thymidine (SHTdR, spec. act. = 6.7 Ci/ mmol, New England Nuclear, Boston, MA) was added and cultures were harvested 4 h later. The effect of removing diamine or DFMO from cultures on the ability of cells to proliferate was also investigated. HDDH or DFMO was included in cultures of spleen cells (2 x 10e/ml) plus mitogen in a total volume of 5 ml. Approximately 24 h later, cell suspensions were mixed and 200 ~l removed and added directly to microtiter wells. The remaining cells were counted and viability was determined both before and after washing in supplemented RPMI plus 5% AB serum. Following 3 washes, 2 × 105 cells were re-cultured in microtiter wells containing mitogen, with or without the same concentration of HDDH or DFMO as in the original culture. All cultures were maintained for an additional 44 h and given a 4 h pulse of 0.5 ~Ci/well SHTdR (total incubation time = 72 h). Ornithine decarboxylase assay Spleen cells (3 × 107) were cultured in 50 ml conical plastic tubes (Corning No. 25339} with Con A (2 ~g/ml) or LPS (20 ~g/ml), with or without DFMO or HDDH, in a total volume of 15 ml. HDDH concentrations ranged from 0.01 to 10.0 mM and DFMO concentrations were 0.01--1.0 mM. Control cultures contained cells and Con A or LPS. After 24 h in culture, mitogen and inhibitor were removed by washing 3 times in Hank's Balanced Salt Solution (GIBCO); cell number and viability were then determined. Cells were resuspended in 1.2 ml of ice-cold 10 mM Tris buffer (pH 7.2) and lysed by 2 cycles of freezing and thawing. Samples were centrifuged at 27 000 x g for 20 min to remove debris and ODC activity in the supernatant was determined by the method of Lau and Slotkin [12]. The incubation medium contained 50 mM pyridoxal-5'-phosphate, 1.5 mM dithiothreitol and 4.5 ~M L[1-14C]ornithine (New England Nuclear, Boston, MA; spec. act. = 53 mCi/mmol). Samples containing 5 mM DFMO served as blanks. Results were recorded as pmol 14C02 evolved/107 cellsPa. 303

Statistical analysis Data are presented as the mean _+ the standard error. Dunnett's t-test was used to evaluate differences between sample means. Viability data were analyzed by linear regression analysis. RESULTS Neutralization of the free amine form of hexamethylene diamine (i.e., HMDA) with HC1 produces the dihydrochloride salt; thus, for all practical purposes, HDDH and HMDA are chemically indistinguishable under the conditions employed in these studies. For this reason, and because experimental results obtained using the commercially prepared salt (i.e., HDDH) or laboratory-neutralized HMDA were identical, results are presented for HDDH only. Trypan blue staining of cultured cells revealed no significant difference between control cultures and those containing HDDH or DFMO plus no mitogen, Con A or LPS (Fig. la). A statistically significant (P < 0.05, r = - 0 . 7 1 6 ) correlation was found between increasing concentrations of DFMO and decreased cell viability for cultures containing PHA (Fig. lb). Proliferative responses to Con A, PHA or LPS in cultures containing HDDH are presented in Figs. 2 a, b and c, respectively. All concentrations of HDDH were suppressive for Con A and PHA stimulated proliferation at 0 and 24 h of culture (P < 0.01). Con-A and PHA-stimulated cultures were less sensitive to HDDH at 48 h and no suppression occurred upon addition at 68 h. LPS stimulated cultures were suppressed at all concentrations of HDDH at 0 h and at > 0.1 mM at 24 and 48 h. Addition at 68 h caused a statistically significant but biologically irrelevant decrease in 3HTdR incorporation at 10.0 mM only. Addition of DFMO to cultures likewise decreased thymidine uptake. Concentrations of 0.1 mM or greater at the initiation of culture or at 24 h significantly (P < 0.01) suppressed responses to both Con A (Fig. 3a) and PHA (Fig. 3b); 0.01 mM DFMO also suppressed the PHA response (P < 0.01) when added at 0 h (Fig. 3b). The Con A response was suppressed only at 1.0 mM DFMO when added at 48 h of incubation whereas the PHA response was not suppressed at 48 h. No effects were observed when DFMO was added at 68 h of incubation. There was a small but statistically significant increase in both the Con A and PHA responses in cultures containing 0.01 mM DFMO at 48 h. Significant decreases (P < 0.01) in 3HTdR uptake occurred at the 1.0 mM concentration in LPS-stimulated cultures to which DFMO was added at 0, 24 and 68 h (Fig. 3c). Cells cultured for 24 h with Con A and HDDH, followed by washing and culturing with Con A only, incorporated 3HTdR in a fashion similar to controls (Fig. 4a). PHA stimulation of washed cells previously cultured with PHA and 2, 4 or 8 mM HDDH induced greater 3HTdR incorporation in these cultures than in cultures originally containing mitogen only (Fig. 4a). The response to LPS was affected only at the highest level of HDDH (i.e., 16

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mM), which decreased tritium incorporation. Significantly decreased responses to all mitogens were obtained when washed cells were recultured with the same concentration of HDDH as in the initial culture {Fig. 4b). Cells initially exposed to 1 mM DFMO plus Con A incorporated more 3HTdR than controls when recultured with Con A only; a similar effect was observed with PHA at 0.1 and 1.0 mM (Fig. 4c). There was an apparent suppression of the LPS response at the lowest concentration of DFMO in the experiment depicted in Fig. 4c, although this was not observed in a subsequent study {data not shown}. Reculture of cells with the highest concentration of DFMO decreased the Con A response whereas the PHA response was not affected at any concentration of DFMO. Reculturing LPS-stimulated cells with 0.01 and 1.0 mM DFMO decreased 3HTdR uptake by approximately 40% (P < 0.01, Fig. 4d). ODC activity of unstimulated cells was not detectable after 24 h in culture. On the other hand, ODC activity in cells cultured for 24 h with Con A rose to 114.0_ 3.0 pmol of 14C0e/107 cells/h or 3.4 _+0.2 pmol ~4C02/107 cells/ h in cells cultured with LPS. For comparison purposes, values obtained for 48 h cultures containing HDDH or DFMO were converted to percent of control, using the above values as 100% for each mitogen and presented in Table I. Percent of control values are also given in Table I for 3HTdR uptake after 48 h of culture with Con A or LPS. Statistical testing of differences between treated and controls were done on actual data. TABLE

I

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OF MITOGEN

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100 ND e ND e 41.5 ± 2.7 d 19.1 ± 0.8 d 3.6 ± 0.2 d

100 1 1 0 . 6 ± 6.7 7 5 . 9 ± 3.5 d 24.6 ± 1.7 d

100 41.2 +_ 3.3 d 13.3 ± 2.2 d 9.8 ± 2.3 d

100 95.3 ± 5.8 51.0 ± 2.8 d 12.0 ± 0.5 d

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Mitogen Fig. 5. Effects of putrescine addition on the proliferative response. (a) Tritium incorporation of spleen cell cultures containing mitogen and putrescine (PUT) (no PUT v / / A , 12.5 ~M PUT k \ \ ~ , 25 ~M PUT ~ ). (b) Cells were cultured with mitogen and HDDH in medium containing no exogenous putrescine ( - P U T ) or at a final concentration of 25 ~M putrescine (+PUT). (1 m M / - P U T [--7--], 1.0 m M / + P U T t \ ], 5.0 m M / - P U T ! / / 2 J , 5.0 mM/ + P U T k \ \ ~ , 10.0 m M / - P U T ~ , , 10.0 m M / + P U T ~ ) . (c) As for 5b, except DFMO was substituted for HDDH (0.01 m M / - P U T v / t , 0.01 m M / + P U T I \ "q, 0.1 raM! - P U T r / / ~ q , 0.1 m M / + P U T [k-'%-~, 1.0 m M / - P U T ~ ¢ ~ ' u , 1.0 m M / + P U T ~ ) . *P < 0.01 versus the response of ceils cultured with the same mitogen plus 25 ~M putrescine only (see panel a).

310

Addition of putrescine (12.5 or 25/~M final concentration) to mitogen stimulated cell cultures had no effect on blastogenesis (Fig. 5a) and did not reverse the inhibitory effects of HDDH on the proliferative response (Fig. 5b, 12.5 pM data not shown). In contrast, both 12.5/~M (data not shown) and 25 pM putrescine were equally effective in reversing the effects of DFMO on the proliferative response (Fig. 5c). DISCUSSION It is well known that polyamines which resemble putrescine (1,4-butane diamine), including hexane diamine [10] suppress ODC activity in a number of in vitro and in vivo systems [reviewed in Refs. 5 and 13] possibly by stimulating the release of a soluble inhibitor of ODC [4,9]. Because ODC activity and polyamine synthesis are required for cellular replication and differentiation, it was of interest to determine whether the time- and dosedependent suppression of lymphoproliferative responses of cultures containing HDDH was the result of interference with polyamine metabolism or had another explanation, including general cytotoxicity. To determine the influence of direct chemical cytotoxicity, trypan blue staining and chemical preincubation were examined. Dye exclusion studies showed no correlation between cell viability and HDDH concentration. Of greater significance, however, was the finding that cells incubated with HDDH proliferated normally in response to mitogens when the chemical was removed. It is thus unlikely that cytotoxicity played a significant role in the observed suppression of lymphoproliferative responses to mitogens. Inclusion of DFMO in cultures was used to establish the effects of blocking ODC activity directly. As with HDDH, time and dose dependent suppression of 3HTdR incorporation was observed and removal of DFMO from mitogen stimulated cultures restored proliferative responses to control levels. Thus, at the levels used in the present study, DFMO inhibited 3HTdR uptake without apparent cytotoxicity. Results of the current study support the view that mitogenic responses of cultured lymphocytes involve the ODC/polyamine system. ODC activity is known to peak within 24 h of mitogen stimulation [7,8] and to remain elevated for at least 48 h [7]. The first 48 h of culture is also the most critical for an optimal response of lymphocytes to mitogens and is characterized by entry of many cells into S phase [14]. Inhibition of polyamine synthesis would thus be expected to have the most marked effect early in the culture period, when the requirement for polyamines is greatest. Our data reflect this temporal relationship: at a given concentration of HDDH or DFMO, delayed addition to cultures diminished the level of suppression obtained. It is also clear that both HDDH and DFMO suppress ODC activity in mitogen-stimulated splenocytes (Table I). The question remains, however, as to whether both HDDH and DFMO suppress mitogenesis exclusively by inhibiting polyamine biosynthesis. Our results strongly suggest that this is the case for DFMO, a well-documented 311

inhibitor of p r e - f o r m e d ODC [11], since addition of m i c r o m o l a r a m o u n t s of p u t r e s c i n e r e s t o r e d t h e p r o l i f e r a t i v e r e s p o n s e . In c o n t r a s t , p o l y a m i n e excess inhibits ODC a c t i v i t y in c u l t u r e d cells b y induction of an inhibitor of ODC s y n t h e s i s r a t h e r t h a n b y d i r e c t l y inhibiting t h e e n z y m e [5,9,13]. In the prese n t study, t h e p a t t e r n of d e c r e a s e d ODC a c t i v i t y and s u p p r e s s e d blastogenesis in H D D H - c o n t a i n i n g c u l t u r e s , which could not be a m e l i o r a t e d b y addition of p u t r e s c i n e , s u g g e s t s t h a t H D D H acts as a p o l y a m i n e (putrescine) analogue, and in effect p r o d u c e s a p o l y a m i n e excess, t h u s d o w n r e g u l a t i n g the a c t i v i t y of ODC. S u p p r e s s i o n of b l a s t o g e n e s i s b y H D D H m a y also involve o t h e r factors distinct f r o m p o l y a m i n e s y n t h e s i s , h o w e v e r , as r e v e a l e d b y the d a t a in T a b l e I. T h a t is, at a c o n c e n t r a t i o n of 0.01 raM, both H D D H and D F M O d e c r e a s e d ODC a c t i v i t y b y a p p r o x i m a t e l y 7 0 % w h e r e a s H D D H , but not DFMO, significantly l o w e r e d t h e r e s p o n s e of T cells to Con A at this c o n c e n t r a t i o n (Table I). T h e s e d a t a s u g g e s t t h a t d e c r e a s e d ODC a c t i v i t y alone is not sufficient to affect l y m p h o c y t e r e s p o n s e s to Con A, although the mechanism(s) u n d e r l y i n g t h e s e d i f f e r e n c e s a r e as y e t unknown. I t could be t h a t H D D H not only induces an inhibitor of ODC activity, b u t also c o m p e t e s w i t h p u t r e s c i n e in m e t a b o l i c e v e n t s n e c e s s a r y for s p e r m i d i n e s y n t h e s i s . R e g a r d l e s s of t h e u l t i m a t e m e c h a n i s m , it is clear f r o m t h e H D D H r e m o v a l / r e c u l t u r e e x p e r i m e n t s t h a t inhibition of p r o l i f e r a t i o n r e q u i r e s the continued p r e s e n c e of H D D H since m i t o g e n r e s p o n s e s of e x p o s e d cells w e r e similar to controls following w a s h i n g and i m m e d i a t e r e c u l t u r e . This s t u d y has s h o w n t h a t H D D H s u p p r e s s e d the r e s p o n s e of cultured s p l e n o c y t e s to m i t o g e n . H D D H m a y act b y i n t e r f e r i n g with intra-cellular p o l y a m i n e m e t a b o l i s m but the m e c h a n i s m of its actions are m o r e complex t h a n simple inhibition of ODC a c t i v i t y and p u t r e s c i n e formation, as was o b s e r v e d for DFMO. REFERENCES 1 A.E. Kulakov, Effect of small concentrations of hexamethylenediamine on experimental animals under conditions of chronic inhalation. Gig. Sanit., 5 (1965) 15. 2 V.M. Shubik, M.A. Nevstruyeva, S.A. Kalnitskii, R.E. Livshits, G.N. Merkushev, E.M. Pilshchik and T.V. Ponomateva, A comparative study of changes in immunological reactivity during prolonged introduction of radioactive and chemical substances into the organism with drinking water. J. Hyg. Epidemiol. Microbiol. Immunol., 22 (1978) 408. 3 S.K. Guha and J. Janne, Inhibition of ornithine decarboxylase in vivo in rat ovary. Biochem. Biophys. Res. Commun., 75 (1977) 136. 4 A.E. Pegg, C. Conover and A. Wrona, Effects of aliphatic diamines on rat liver ornithine decarboxylase activity. Biochem. J., 170 (1978) 651. 5 A.E. Pegg and P.P. McCann, Polyamine metabolism and function. Am. J. Physiol., 243 (Cell Physiol. 12) (1982) C212. 6 D.H. Russell, C.V. Byus and C.A. Manen, Proposed model of major sequential biochemical events of a trophic response. Life Sci., 19 (1976) 1297. 7 J.E. Kay and A. Cooke, Ornithine decarboxylase and ribosomal RNA synthesis during the stimulation of lymphocytes by phytohaemagglutinin. FEBS Lett., 16 (197119. 8 I.G. Scott, H. Poso, K.E.O. Akerman and L.C. Andersson, Rapid activation of ornithine decarboxylase by mitogenic (but not by nonmitogenic) ligands in human T lymphocytes. Eur. J. Immunol., 15 (1985) 783.

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