Pharmacological inhibition of interleukin-1 activity on T cells by hydrocortisone, cyclosporine, prostaglandins, and cyclic nucleotides

lmmunopharmacology, 15 (1988) 47-62 Elsevier

47

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Pharmacological inhibition of interleukin-1 activity on T cells by hydrocortisone, cyclosporine, prostaglandins, and cyclic nucleotides Daniel E. Tracey, M a r i l y n M. H a r d e e , K a r e n A. R i c h a r d and Jeff W. Paslay ttypersensitiviO, Diseases Research. The Up/ohn Company, Kalamazoo, MI 49001, U.S.A. (Received 23 April 1987: accepted 29 October 1987)

Abstract: The effects of a panel of hormones and pharmacological agents on the activation of T cells by a combination of interleukin-1 and phytohemagglutinin (IL-I/PHA) was studied. Pharmacological effects on various stages of IL-l/PHA-induced interleukin-2 (IL-2) production by the cloned murine thymoma cell line LBRM-33-1A5.7 were dissected using a multi-step assay procedure. A 4-h lag phase in the kinetics of IL-2 production allowed the operational definition of an early, IL-l-dependent programming stage, followed by an IL-2-production stage of the assay. A cell-washing procedure between these stages was introduced in order to distinguish |L-1 receptor antagonists from functional I L-1/PHA antagonists. Hydrocortisone and cyclosporine were potent inhibitors (active in the nM range) of both stages of IL-2 production, suggesting that neither is an |L-I receptor antagonist. The cyclic adenosine monophosphate (cAMP)elevating agents prostaglandin Ez, dibutyryl cAMP, and theophylline inhibited IL-2 production during the early, IL-l-dependent programming stage. By contrast, prostaglandin F2~ and dibutyryl cyclic guanosinc monophosphale did not appreciably inhibit IL1/PHA activity. These results are discussed in relationship to the effects of these test agents in thymocytc I L-I assays or mitogenesis assays and the implications toward understanding the mechanisms underlying IL-I/PHA activation of T cells.

Key words: Interleukin-l: Interleukin-2; T cells: Hydrocortisone; Cyclosporine: Prostaglandins; Cyclic nucleotides

Introduction

Activation of T lymphocytes by antigens or mitogens is greatly enhanced by the cytokine interleukin-1 (IL-1) (Gery et al., 1972; Durum et al., 1985). IL-1 molecules bind to specific receptors on the surface of T cells and other cells (Dower et al., 1985). Murine thymocytes stimulated with IL-1 and a Tcell mitogen such as phytohemagglutinin (PHA) Correspondence: D.E. Tracey, Hypersensitivity Diseases Research, The Upjohn Company, Kalamazoo, M1 49001, U.S.A. Abbreviation,s'." IL, interleukin: HC, hydrocortisone; CS, cyclosporine; PG, prostaglandin; DBcAMP, dibutyroyl cyclic adenosine monophosphate; DBcGMP, dibutyryl cyclic guanosine monophosphate: PHA, phytohemagglutinin; 1A5.7, LBRM-33-1A5.7 cells; FCS, fetal ca!f serum; DMSO, dimethylsulfoxide; 1C5o, concentration for 50% inhibition.

have been used extensively as a model of ILl/antigen-induced T-cell activation. It has been postulated that IL-1 and PHA act synergistically to induce production of interleukin-2 (IL-2) by, and enhance IL-2-receptor expression on, mature thymocytes and T lymphocytes (Larsson et al., 1980). The secreted IL-2 then interacts with IL-2 receptors on some T cells, causing them to proliferate (Smith and Ruscetti, 1981). However, it has been very difficult to clearly evaluate the mechanism of IL-1/mitogen costimulation of T cells due to the heterogeneous nature of these cells and the multiplicity of cellular events occurring during this process. More recently, several cloned T lymphoma or T cell lines have been described which can be induced by IL-1 and mitogens to express increased levels of

0162-3109/88/$03.50 (~') 1988 Elsevier Science Publishers B.V. (Biomedical Division)

48 IL-2 receptors and to produce IL-2 (Gillis et al., 1980; Kaye and Janeway, 1984). One such thymoma cell line, LBRM-33-1A5, expresses several hundred IL-I receptors per cell (Dower et al., 1985) and responds to IL-I plus PHA, or IL-1 plus the ionophore A23187 (Zlotnick and Daine, 1986), by producing IL-2 and increasing IL-2 receptor expression. Not only are these cells much more sensitive to IL-1 than murine thymocytes, but the cloned line provides a homogeneous cell population with which to study the mechanism of T-cell activation by IL-I. Natural hormones and pharmacological agents have been used in the thymocyte costimulator assay to probe the mechanism and regulation of IL-1 stimulation of T cells. Glucocorticosteroids and the immunosuppressive fungal metabolite cyclosporine (CS) are known to inhibit thymocyte proliferation in IL-1/mitogen costimulator assays (Gery et al., 1983). However, the mechanisms by which these agents inhibit these assays are not well understood. Inhibition could occur at the level of IL-1 or mitogen receptors, signal transduction leading to IL2 biosynthesis, IL-2 secretion, IL-2 receptor expression, and/or cell proliferation in response to IL-2. In order to gain a better understanding of the mechanism of action of these agents on IL-l-induced T-cell activation, we have evaluated hydrocortisone (HC), CS, PGE2, and several cAMP-elevating agents on cloned thymoma cell lines. We report here the effects of these agents on IL-l-induced IL-2 production by LBRM-33-1A5 cells and on IL-2-induced proliferation of HT-2 cells. In addition to dissecting the effects of these agents on IL-2 production and IL-2-induced proliferation, we report a modification of the I LI/LBRM-33-1A5 assay to allow for evaluation of the ability of test agents to act as IL-l-receptor antagonists. Materials and Methods

Cell lines The routine T cell line LBRM-33-1A5, originally described by Gillis et al. (1980), was obtained from

the American Type Culture Collection (Rockville, MD) and was further subcloned. One subclone, LBRM-33-1A5.7, hereafter referred to as 1A5.7, was maintained at 37°C in a 5% CO2 atmosphere in culture medium: RPMI-1640 medium supplemented with 100 U/ml penicillin, 100/zg/ml streptomycin (GIBCO, Grand Island, NY), and 10% heat-inactivated fetal calf serum (FCS; Sterile Systems, Logan, UT). The IL-2-dependent murine T cell line HT-2 was obtained from G. Fatham (Stanford University). These cells were maintained by continuous culture in culture medium further supplemented with 10 mM HEPES buffer (GIBCO), 5 x 10 s M 2-mercaptoethanol and 15% supernatant from concanavalin A-stimulated rat spleen cells. This supernatant, used as a source of lk-2, was generated by stimulation of Sprague-Dawley rat spleen cells at 5 x 106 cells/ml in culture medium with 5/ag/ml concanavalin A (Pharmacia, Piscataway, NY) for 48 h at 37°C. Reagents and test compounds Murine IL-I was prepared from murine P388D~ cells (American Type Culture Collection, Rockville, MD) stimulated with 10 -s M mezerein (LC Services, Wolburn, MA) as described previously (Mizel, 1982). Pooled supernatants were concentrated by precipitation with 65% saturated (NH4)2SO,, and ultrafiltration using a YM-10 membrane (Amicon, Danvers, MA) and fractionated by chromatography on Biogel P-30 (Biorad, Richmond, CA). Fractions comprising the major peak ( ~ 17 kDa) were pooled. The IL-1 content in this preparation was measured as 150 U/ml by titration in a murine thymocyte costimulator assay (Gery et al., 1972), wherein half-maximal activity was defined at 1 U/ml. PHA was obtained from Burroughs-Wellcome (Wellcome Reagents Division, Research Triangle park, NC) as purified PHA (Lot HA-17). Test compounds were prepared as 0.2 or 0.02 M stock solutions in dimethylsulfoxide (DMSO; Burdick and Jackson, Muskegon, MI). CS was a gift from Sandoz (Basel, Switzerland). HC, dibutyryl cAMP (N ~', OZ'-dibutyryladenosine Y:5'-cyclic monophosphor-

49 ic acid), dibutyryl cyclic GMP (N 2, O2~-dibutyryl guanosine 3',5'-cyclic monophosphate), and theophylline were purchased from Sigma (St. Louis, MO). Prostaglandin E2 (PGE2) and PGF2, (THAM salt) were synthesized at The Upjohn Company. IL-I assays The assay procedures used to evaluate drug effects on IL-1 activity were based on the assay originally described by Gillis and Mizel (1981). This assay was adapted for use in the evaluation of IL-1 modulatory compounds by adding a half-maximal amount ofmurine IL-1 to a mixture of 1 x 105 1A5.7 cells, 2 #g/ml PHA and aliquots of solutions of test compounds in quadruplicate in wells of V-bottomed microtiter plates (NUNC-96V; GIBCO). Each component of the assay mixture was prepared in IL-1 assay medium: RPMI-1640 medium supplemented with 5% FCS, 50 #g/ml gentamicin (GIBCO) and 20 mM HEPES buffer. The plates were incubated at 37°C for 4 h, centrifuged at 300 x g for 6 min and the cell supernatants were vacuum aspirated using a Dynatech 12-channel manifold (Dynatech, Alexandria, VA). The cell pellets were washed twice by resuspension with 200 #1 phosphate-buffered saline (PBS; GIBCO) containing 1% FCS, followed by centrifugation and supernatant aspiration. The washed cell pellets were resuspended in 200 #1 IL-1 assay medium containing 2 #g/ml PHA with or without fresh aliquots of test compounds. The plates were then incubated for 20 h at 37°C, centrifuged, and 100 #1 aliquots of the cellfree supernatants were removed for measurement of IL-2 content. 1L-2 assays Cultures of HT-2 cells were centrifuged and washed three times in IL-2 assay medium: RPMI-1640 medium supplemented with 50 #g/ml gentamicin, 2% FCS, 20 mM HEPES buffer and 5 x 10 _5 M 2mercaptoethanol (Eastman Kodak, Rochester, NY). Fifty microliters of HT-2 cells at 2 x 105 cells/ml in IL-2 assay medium were dispensed into wells of a flat-bottomed 96-well microtiter plate

(Falcon 3075). The 100 #l supernatants from the IL-1 assays were then added to the HT-2 cells. Finally, 50 pl of IL-2 assay medium or a half-maximal amount of IL-2 (rat concanavalin A spleen cell supernatant) were added to the wells. The plates were then incubated at 37°C for 20 h. To each well 50 #1 [methyl-3H]thymidine (2 Ci/mmol; Amersham, Arlington Heights, IL) diluted to 10 #Ci/ml in IL2 assay medium were added. After 4 h at 37°C the contents of each well were transferred to glass-fiber filters using a Skatron Cell Harvester (Skatron, Sterling, VA). The filters were placed in plastic vials with 3 ml ACS (Amersham) scintillation fluid and were counted in a Beckman liquid scintillation counter equipped with a radioimmunoassay software program. Levels of IL-2 were calibrated to the NIH Biological Response Modifiers program standard IL-2 preparation obtained from J. Talmadge (National Cancer Institute). Toxicity assays Each test compound was evaluated for the degree of toxicity to 1A5.7 cells imparted during the 20-h incubation period in the absence of IL-I. After supernatants were removed for IL-2 assay, the viability of drug-treated 1A5.7 cells was assessed with a colorimetric assay of mitochondrial activity. This MTT toxicity assay (Chemicon International, El Segundo, CA) depends on the conversion of the yellow substrate MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide to a dark blue formazan product (Mossman, 1983). The 1A5.7 cells were mixed with MTT, transferred to flat-bottomed NUNC Immulon ELISA plates and incubated for 3 h at 37°C. The formazan product was dissolved by the addition of 200 #1 isopropanol/HCl and the optical density in each well was read at 550 nm in a Titertek Multiskan spectrophotometer (Flow Laboratories, McLean, VA). Percent toxicity was calculated relative to control wells in which 1A5.7 cells were cultured with IL-1 assay medium alone. That this MTT assay reliably measures cell viability has been documented (Mossman, 1983) and confirmed in our laboratory in comparison with trypan blue due exclusion assays.

50

Drug~treatment protocols Using the basic assay protocols described above, four different modifications were employed for each test compound. These protocols are illustrated in Fig. 1 and consist of drug treatments of IA5.7 cells in the early (0 4-h 'programming') and late (4-24h 'IL-2 production') stages of the IL-1 assay. Two protocols, A and C, represent drug treatment in these stages in the presence of IL-I from 0-4 h. Protocols B and D are controls done in the absence of IL-1 to measure the effects of drug carried over to the HT-2 cells in the IL-2 assay in the presence of exogenously added IL-2. The data are expressed relative to the drug concentrations in the IL-I portions of the protocols, but it should be noted that the actual drug concentrations in the IL-2 assays were half those in the corresponding 1L-I portions. The drug carryover controls do not strictly measure the effects of test drugs on IL-2 activity since they 4H PROGRAMMING

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also control for drug degradation, drug metabolism by IA5.7 cells, drug interaction with PHA, and other cell-derived factors induced by the drugs during the incubation with IA5.7 cells. In some experiments a filth protocol was used in which test drugs were present throughout the entire 0 24-h IL-1 assay as well as being carried over into the IL-2 assay. Exogenous, half-maximal amounts of 1L-I or IL-2 were added to the assays at the beginning of the IL-1 or 1L-2 assays, respectively.

Calculations and statistical analyses In each experiment the mean levels of IL-1 or 1L-2 activity in quadruplicate wells were quantified using standard IL-1 and IL-2 curves plotted as weighted linear regression analyses of the log I L-I or I L-2 concentration versus a logit transformation of the percent of maximum pH]thymidine incorporation of the IL-I or IL-2 standards. Each plate contained six sets of quadruplicate control wells containing the same amounts of D M S O as in wells with test drugs. The effective concentrations of IL-1 and I L-2 in the presence of test drugs were calculated as a percent of the mean control IL-I or 1L-2 wells on each plate. Each test compound was evaluated in 2 8 separate experiments at each of several concentrations. At any given drug concentration, the means and standard errors of the means of the percents of control IL-1 or IL-2 activities were calculated. Statistical analyses of the differences between drug treatments and controls were based on one-way analyses of variance of the percents of control in all 2-8 experiments with a given concentration of drug.

Results

IL-1 stimulation q]" 1A5 cells Stimulation of 1A5,7 cells with IL-1 and PHA induces the de novo synthesis and release of IL-2 within 24 h (Gillis and Mizel, 1981). In order to design drug-treatment protocols to probe the early and late stages of IL-2 production, the kinetics of

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IL-2 production were measured. As shown in Fig. 2, an optimal concentration of IL-1 (0.15 U/ml) induced detectable IL-2 in the supernatants 6 h, but not 4 h, after addition. Maximal IL-2 production occurred after 24 h incubation with IL-1. No IL-2 production by 24 h was seen in the absence of ILl (data not shown). Since no IL-2 could be detected up to 4 h after IL-1 addition, the 0-4-h period was selected as the early or 'programming' stage of IL-2 production. Two cell washes at the 4-h point serve to remove non-cell-associated IL-1 (and test drug in later experiments). Despite this removal of IL-1, IL-2 production continued in the presence of P H A alone. Fig. 2 shows that, regardless of the concentration of IL-1 present from 0 4 h, IL-2 production began at 6-8 h and was maximal at 18 24 h. About twice as much IL-1 was required in the 0-4-h stage with a wash step at 4 h, as compared with no wash, to yield a similar level of IL-2 production at 24 h. As seen most dramatically in Fig. 2 with the highest dose of IL-1, an apparent decrease in IL-2 level in the supernatant was evident between the 24- and 30-h time points, but only in washed cultures. If

this decrease in supernatant IL-2 content is due to IL-2 adsorption or catabolism in the cultures, these data may indicate that IL-2 production, initiated by IL-1 being present only from 0-4 h, ceases by 24 h of culture. Addition of IL-1 to cultures after a 4-h preincubation with PHA resulted in somewhat lower IL-2 levels at 18 24 h than when IL-1 and PHA were both present during the preincubation step (data not shown). In any case~ the kinetics of IL-2 production by IL-1/PHA-stimulated 1A5.7 cells shown in Fig. 2 enable us to operationally define the 0-4-h period as the IL-2 programming stage and the 4 24-h period as the IL-2-production stage, with or without a wash step at 4 h. Strictly speaking, however, what is called 'IL-2 production' is really the total accumulated IL-2 in the cultures at 24 h. As shown in Fig. 2, this level may be an underestimate if IL2 catabolism or adsorption occurs during this time.

Overall effects of test drugs on IL-1 and IL-2 activities Prior to a dissection of drug effects in the early and late stages of the IL-1 assay, the test drugs were added to 1A5.7 cells for the entire 0 24-h culture period, without a wash step at 4 h. Fig. 3 shows the percents of control IL-1 and 1L-2 activities in the presence of test drugs at various concentrations between 10 3 M and 10- lO M. The IL-2 panel shows the carryover effects of the drugs onto the HT-2 cells in the IL-2 portion of the assay. The only statistically significant (p < 0.05) inhibition of 1L-2driven HT-2 cell proliferation at non-toxic ( < 20% toxicity) drug concentrations was seen with PGE2 at 10 ~ to 10 -8 M, PGF2~ at 10 _5 M, D B c A M P at 10- 4 M, and with theophylline at 10- 3 M. In contrast, all but one drug, DBcGMP, gave significant inhibition in the IL-1 assay. Significant inhibition of IL-1 activity without toxicity was seen with HC at 10 -8 M, C S a t 10 -8 M and 10 9 M, PGE2 at 10 5 M t o 10 l o M , PGF2, at 10 - S M , D B c A M P at 10 4 M and 10 5 M, and theophylline at 10 3 M. Precise quantitative evaluation of these 0-24-h drug-treatment data is complicated by the toxic

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effects of the test drugs and by the carryover effects of the test drugs in the IL-2 assay. Nevertheless, Fig. 3 shows that H C and CS selectively inhibited IL-l-induced IL-2 production at low concentrations. In addition, these data suggest that PGE2, DBcAMP, and theophylline may be relatively weak inhibitors of IL-l-induced IL-2 production, independent of their inhibitory effects on IL-2-driven HT-2 cell proliferation. The weak IL-1 inhibition seen with PGF2~ at 10 -5 M appears to be accounted for entirely by inhibition in the HT-2 cell assay.

Effects of test drugs on d(fferent stages (~[ the IL-1 assay In order to further evaluate the effects of test drugs on different stages of the 1L-1 and I L-2 assays, each drug was tested in 3-8 separate experiments, each consisting of the four drug-treatment protocols diagrammed in Fig. 1.

The results obtained with H C are shown in Fig. 4. As seen in the left panel ('IL-I'), strong inhibition of IL-1 activity occurred at 10 -8 M or higher concentrations of H C present during the 0 4-h programming stage. The drug carryover control which contained no IL-1 and to which exogenous IL-2 was added (left panel, 'IL-2'), shows that none of the effects of H C in the 0-4-h stage of the IL-1 assay were due to incomplete removal of the drug in the wash step. As seen in the right panel ('IL-2'), H C deliberately carried over into the HT-2 cell assay was a weak inhibitor ( ~ 2 0 % ) of IL-2-driven HT2 cell proliferation at the highest non-toxic concentration tested, 10 s M. However, 10 8 M H C present from 4 24 h, following IL-I stimulation of 1A5.7 cells (right panel, ' I L - I ' ) was markedly inhibitory for IL-2 production. Assuming that up to 20% of this inhibition was due to drug carryover into the HT-2 cell assay, these studies show that the IC5o for H C in the 4 24-h IL-l-dependent IL-2-

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Fig. 4. Effects of HC on IL-I and IL-2 activities when tested as described in Fig. 1. Left panel: percent of control IL-2 production ('IL-I') or activity ('IL-2') following drug treatment of IA5 cells from 0-4 h in the presence of 2 ,ug/ml PHA and 0.05 U/ml IL-1 (0) or no IL-1 (©). In the latter case, I U/ml IL-2 was added to the 1A5 cell supernatants at 24 h. Right panel: as in left panel except that the drug was present only after the 4-h wash step, from 4-24 h. Asterisks denote > 20% toxicity of drug to 1AS cells measured at 24 h in the absence of IL-I. The average toxicity value in these experiments was 43% at 10-7 M HC present from 4-24 h. Statistically significant inhibition (p < 0.05) was seen at the following points: left panel, 'IL-I' at 10 s, 10-v and 10 6 M ; right panel, 'IL-I' at 10-s and 10 7 M, 'IL-2' at 10 8 and 10_7 M HC. The data in this figure are the means and standard errors of eight separate experiments.

p r o d u c t i o n stage was < 10 - s M, roughly c o m p a r able to the drug's effect in the 0~4-h p r o g r a m m i n g stage. H C was far more toxic to 1A5.7 cells when present from 4-24 h t h a n from 0-4 h, however. Similar studies with CS are s u m m a r i z e d in Fig. 5. This c o m p o u n d was n o t toxic to 1A5.7 cells at c o n c e n t r a t i o n s u p to 10 - v M. P o t e n t i n h i b i t i o n of I L - l - i n d u c e d IL-2 p r o d u c t i o n was seen at 10 - s M a n d 10- 7 M, regardless of the period d u r i n g which the drug was present in the assay. D r u g carryover

controls showed that CS was only moderately inhibitory ( ~ 3 0 % ) at 10 _7 M on IL-2-driven H T - 2 cell proliferation (right panel, 'IL-2'), a concentration at which 100% i n h i b i t i o n of IL-1 activity was seen in both stages o f the assay. These data indicate that the IC5o for CS in the 0 - 4 - h p r o g r a m m i n g stage was slightly lower ( ~ 4 × 10 -9 M) t h a n in the 4 24-h I L - 2 - p r o d u c t i o n stage ( ~ 9 × 10 -9 M). Fig. 6 shows the results with PGE2 in the different stages o f the IL-1 assay. W h e n PGE2 was

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Fig. 5. Effect of CS on 1L-1 and I L-2 activities when tested as described in Fig. 1. All procedural details are as described in Fig. 4. No toxicity was observed. Statistically significant (p < 0.05) inhibition was seen: left panel, 'IL-I" at 10 ~ and l0 -~ M: right panel, 'IL-I' at 10 s and 10 7 M, "IL-2" at 10 7 M CS. The data m this figure are the means and standard errors of eight separate experiments.

present d u r i n g the 0 - 4 h o u r period, strong inhibition was seen at 10 -5 M a n d 10 4 M, a l t h o u g h some o f this inhibition was due to d r u g c a r r y o v e r into the IL-2 assay (left panel. "IL-2'). The carryover m a y be due to i n c o m p l e t e washing a n d / o r release o f cell-associated P G E 2 after the wash step. T a k i n g the d r u g c a r r y o v e r c o n t r o l into c o n s i d e r a tion, the ICso for PGE2 in the 0 4-h p r o g r a m m i n g stage was a p p r o x i m a t e l y 5 x 10 6 M. The reason that d r u g c a r r y o v e r h a d such an i n h i b i t o r y effect is shown in the right panel ( ' I L - 2 ' ) , wherein P G E 2 deliberately carried over from the 4-24-h t r e a t m e n t significantly inhibited IL-2-driven H T - 2 cell proliferation, even at 10 v M, the lowest c o n c e n t r a t i o n shown. F u r t h e r dilutions showed that this inhibition persisted at 10 s M. but not at 10 ~) M or

10 ~o M ( d a t a n o t shown). A l t h o u g h the inhibition o f PGE2 from 4 24 h following I k - I s t i m u l a t i o n (right panel, ' I L - l ' ) was significantly m o r e p r o n o u n c e d than in the d r u g c a r r y o v e r c o n t r o l at 10 (~ and 10 s M, an 1Cso value for the effect o f PGE2 on the I L - 2 - p r o d u c t i o n stage o f the I L-I assay could not be estimated because o f its i n h i b i t o r y activity in the IL-2 assay. Significant 1A5.7 cell toxicity was o b s e r v e d when 10 -a M P G E 2 was present d u r i n g the 4-24-h period. In c o n t r a s t to the effects o f PGE2, PGF2~ did not significantly inhibit I L- 1 activity when present during the 0 4-h p e r i o d at c o n c e n t r a t i o n s up to 10 .s M (Fig. 7). Significant inhibition ( ~ 6 0 % ) o f I L - I induced 1L-2 p r o d u c t i o n was seen, however, when 10 s M PGF2~ was present d u r i n g the 4 24 h

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Fig. 6. Effects of PGE2 on IL-I and IL-2 activities when tested as described in Fig. 1. All procedural details are as described in Fig. 4. The average toxicity (*) to 1A5 cells at 10 -4 M PGEz present from 4-24 h was 32%. Statistically significant (p < 0.05) inhibition was seen: left panel, q L - l ' at 10 -5 and 10 _4 M, 'IL-2' at 10 -5 and 10 -4 M; right panel, all concentrations of PGEz in both 'IL-I' and qL-2' curves. Statistically significant (p < 0.05) differences between the "IL-I' and qL-2' values were seen: left panel, 10 - s and |0 -4 M; right panel, 10 -6 and 10 5 M PGE2. The data in this figure are the means and standard errors of five separate experiments.

period. Part of this inhibition was due to the inhibitory effect ( ~ 2 5 % ) of the carryover of 10 -5 M PGF2~ on IL-2-driven HT-2 cell proliferation. These results indicate that PGF2, was a very weak inhibitor of IL-l-induced IL-2 production, acting during the IL-2-production stage at an IC5o greater than 10 -5 M. As shown in Fig. 8, dibutyryl cAMP was strongly inhibitory for IL-l-induced IL-2 production when present in the 0 4-h programming stage at 10 4 or 10 3 M. None of this effect was due to drug carryover into the IL-2 assay. However, DBcAMP was quite inhibitory at 10 - 4 M and 10 3 M (toxic) when deliberately carried over from the 4-24-h treatment period into the HT-2 assay (right panel,

'IL-2'). Similar degrees of inhibition were seen at all concentrations tested for DBcAMP in the 424-h period in the presence or absence of IL-1. This suggests that DBcAMP had little or no effect on IL-2 production when present from 4 24 h in the IL-1 assay. Dibutyryl c G M P inhibited lL-l-induced IL-2 production only when present from 0-4 h at 10 3 M (Fig. 9). The slight inhibitory activity ( ~ 2 5 % ) of D B c G M P on IL-2-driven HT-2 cell proliferation at 10-3 M accounted for the apparent inhibition in the 4 24-h stage (right panel). Inhibition of IL-l-induced IL-2 production was also seen with theophylline present from 0~4 h at 10 -4 and 10 - 3 M (Fig. 10). No drug carryover

56 PROSTAGLANDIN

F2~

120 •

120 -

100

100

80

(~

60

-

i

I....

80-

60o

40

40 UA

IL-I

(.)

,,=,

20

20

(l.

o

LOG DRUG CONCENTRATION (M)

/

]

0 4H 24H 0 4H 24H Fig. 7. Effects of PGF2~ on IL-I and IL-2 activities when tested as described in Fig. I. All procedural details are as described in Fig. 4. No toxicity to 1A5 cells was observed. Statistically significant inhibition (p < 0.05) was seen: right panel, "IL-I' 10-5 M PGFz~, Statistically significant (p < 0.05) differences between qL-l' and "IL-2"curves were seen only in the right panel at 10-5 M PGF2=. The data in this figure represent the means and standard errors of three separate experiments.

effects were seen, and the ICso for theophylline inhibition in the p r o g r a m m i n g stage was approximately 10 4 M. Slight ( ~ 2 0 % ) inhibition o f IL2-driven H T - 2 cell proliferation was seen with 10 -4 M theophylline carried over from the 4-24-h treatment. The 10 -3 M concentration was toxic to 1A5.7 cells during the 4-24-h period. N o difference between the I L - I and IL-2 curves in the right panel was seen, suggesting that theophylline did not inhibit I L - l - i n d u c e d IL-2 p r o d u c t i o n during the 4 24-h stage. A s u m m a r y o f the effects on I L - l - i n d u c e d IL-2 production by each o f the seven test drugs is given in Table I. Wherever possible, attempts were made to estimate an ICso value for each drug in each stage o f the assay by allowing for the effects o f drug

carried over into the I L-2 assay. The only drug with no calculable activity was PGF2=. The active drugs fell into three categories: those that potently inhibited both the p r o g r a m m i n g and IL-2-production stages (HC and CS), those that primarily inhibited the p r o g r a m m i n g stage at moderate concentrations (PGE2, D B c A M P ) , and those that primarily inhibited the p r o g r a m m i n g stage at very high concentrations ( D B c G M P , theophylline).

Discussion

We have two main objectives in using T cells lines for the pharmacological evaluation o f I L- 1 activity: to probe the biochemical mechanisms involved in

57

DIBUTYRYL CYCLIC AMP 120.

120 1

100

Ill

~

IL-2

z O a.

~

¢v"

80

..J

O

N

60

o

40

z O

40

p-

IL-I~ I~ IL-2

z

~IL-1

LU

20

~

20

D.

)*

-5

1 0

-4

-3 LOG DRUG CONCENTRATION (M)

] 4H

24H

0

4H

24H

Fig. 8. Effects of DBcAMP on IL-I and 1L-2 activities when tested as described in Fig. 1. All procedural details were as described Fig. 4. The average toxicity (*) to 1A5.7 cells at 10 4 M DBcAMP present from 4 24 hours was 40%. Statistically significant (p 0.05) inhibition was seen: left panel 'IL-I' 10 -4 and 10 -3 M; right panel, 'IL-I' at 10 -s. 10 -4, 10 -3 M; 'IL-2' at. 10 -4 and 10 -~ DBcAMP. Statistically significant (p < 0.05) differences between 'IL-I' and 'IL-2' curves were seen: left panel, 10 -~ and 10 3 DBcAMP. The data in this figure are the means and standard errors of two separate experiments.

IL-1 activation of T cells and to provide a foundation for the search for new agents which selectively modulate IL-1 bioactivities. The present study has provided information in both these directions. The modified 1A5.7/HT-2 multi-step IL-1 assay has enabled us to identify the temporal and cellular sites of action of a panel of pharmacological agents. Such a dissection of the sites of drug action could not have been achieved in a standard thymocyte assay, because of the heterogeneous nature of this cell population. However, the 1A5.7 cells used in our studies demonstrated the same kinetics of IL-2 production as mitogen- or antigenstimulated murine lymphocytes (Gillis et al., 1978). The 4-6-h lag period for IL-1/PHA-induced IL-2

in < M M

secretion by 1A5 cells was the basis for an operational definition of an IL-1/PHA-programming stage (0~4h) and an IL-2-production stage (4 24 h) of the IL-l-dependent phase of the assay. In addition, effects of drug carryover to the lL-2-dependent HT-2 cell-proliferation stage could be distinguished from the IL-l-dependent effects. IL-1 binding to LBRM-33-1A5 cells has been shown to occur rapidly at 37°C, to be of high affinity, and to be followed by rapid (less than 2 h) internalization within the cell (Dower et al., 1985; Lowenthal and MacDonald, 1986). Therefore, drugs which inhibit the IL-1 assay during the IL2-production stage, after exogenous IL-1 has been washed away, would be unlikely to act as IL-l-re-

58 DIBUTYRYL C Y C L I C GMP

120"

120

/~

I IL-2

100

Ik-2

80

8O

60

a0

Z

40

20

iL_l l

Z O U-

o

I--Z UJ

40

20 o.

o

_;

0

4H

o LOGDRUGCONCENTRATION (M) 24H

0

4H

_;

24H

Fig. 9. Effects of DBcGMP on IL-I and IL-2 activities when tested as describcd in Fig. 1. All procedural details are as described in Fig. 4. No toxicity to 1A5.7 cells was observed. Statistically significant (p < 0.05) inhibition was seen: left panel, "IL-I" at 10-3 M; right panel, 'IL-I" and "IL-2" at 10 3 M DBcGMP. The data in this figure are the means and standard errors of two separate experiments at 10 3 M DBcGMP, and a single experiment for the lower concentrations,

ceptor antagonists since the 1L-l-bound receptors have been internalized and IL-2 p r o g r a m m i n g has been initiated during the first 4 h. We would expect an I L - l - r e c e p t o r antagonist to inhibit primarily during the I L - 1 / P H A - p r o g r a m m i n g stage, but inhibition in this stage alone certainly does not prove that an agent is an I L - l - r e c e p t o r antagonist. Interestingly, H C and CS had similar effects and potencies in these assays. CS was a b o u t twice as potent as H C on I L - l - d e p e n d e n t activity, and was far less toxic to 1A5.7 cells than HC. Both compounds were active in the nM range in both the p r o g r a m m i n g and IL-2-production stages. Carryover o f neither c o m p o u n d appreciably inhibited IL-2-dependent proliferative activity. Since both c o m p o u n d s were potent inhibitors of the IL-2-pro-

duction stage when added to the 1A5.7 cells after |L-1 and P H A had triggered their receptors, neither H C nor CS could be I L - l - r e c e p t o r antagonists. The ICso for both c o m p o u n d s in each o f the IL-l-dependent stages was comparable to the ICso when drug was present for the entire 0 24-h period on 1A5.7 cells (Table I). This suggests that these compounds block one or more steps in IL-1 a n d / o r P H A signal transduction, IL-2 m R N A synthesis, IL-2 protein synthesis, or IL-2 secretion. Our results with H C confirm and extend previous observations that H C or dexamethasone inhibit IL-1- or I L - 1 / P H A - i n d u c e d thymocyte proliferation (Gery et al., 1983), T-cell mitogenesis (Gillis et al., 1979), and autologous M L R assays (Palacios and Sugawara, 1982). In the latter two cases, inhi-

59

THEOPHYLLINE 120

120IL-2

100

tu

100

-

-

B

.

oo Z O et

~

80

80

...1

0

N

60

Z

60

o u.

40

o

40

I-Z w

"" w

20

20

o.

0 -5

-

-3

LOG DRUG CONCENTRATION (M)

0

4H

24H

0

4H

24H

Fig. 10. Effects of theophylline on IL-I and lL-2 activities when tested as described in Fig. 1. All procedural details were as described in Fig. 4. The average toxicity (*) to 1A5.7 cells at 10 -3 M theophylline present from 4-24 h was 20%. Statistically significant (p < 0.05) inhibition was seen: left panel, ' I L - I ' at 10 -4 and 10 _3 M: right panel, "IL-l" at 10 3 M, ' I L - T at 10 -4 and 10 3 M theophylline. The data in this figure represent the means and standard errors of two separate experiments.

bition was shown to be at the level of IL-2 production, not at the level of IL-2-induced T-cell proliferation. However, in the IL-1-dependent thymocyte assay, dissection of the level at which inhibition occurred was not attempted. The mechanisms by which glucocorticoids inhibit IL-2 production are not known, but cAMP elevation is probably not involved (Gehring and Coffino, 1977). Glucocorticoids bind to intracellular receptors and send signals to the nucleus to initiate gene transcription for proteins, including lipocortins (Claman, 1983). We are currently investigating the role of lipocortins in steroid-induced inhibition of IL-2 production. Glucocorticoids also activate endonucleases, particu-

larly in rodent lymphocytes (Claman, 1983), which explains the high levels of toxicity we observed with HC on 1A5.7 T cells. This complicates the interpretation of the mechanism of HC inhibition of IL-2 production since the concentration window between inhibition and toxicity is quite narrow with glucocorticoids. Furthermore, it is paradoxical that despite the IL-l-inhibitory effects of glucocorticoids, a recent report indicates that such compounds increase IL-l-receptor expression on lymphoid cells and cell lines (Akahoshi et al., 1987). These results may be interpreted as further evidence that glucocorticoids do not block IL-1 activity at the lk-I receptor level.

60 TABLE 1 Summary of the clt'ects of test compounds on 1L- I,'PHA and 1L- I/PHA and 1L-2 activities Test compound

1C5{} (HM) l\~r IL-1/PHA activity"

1C~o {HM) for IL-2 activityb

0-24h

0 4h

4 24h

HC CS PGE2

~0.006 ~ 0.003 NE d

-0.010 ~ 0.004 ~8

~0.010 ~ 0.009 NE

NF NI ~5

PGF2~ DBcAMP DBcGMP

N1 ~ 100 NI

NI ~ 20 ~ 600

NE NE NI

NI ~ 50 N1

Theophylline

NE

~ 200

NE

NE

~' Data from Figs, 3 10 were evaluated for inhibition of I L - I / P H A induced IL-2 production when drug was present during each of the three time periods shown. h Data from Figs. 3-10 were evaluated for inhibition of IL-2 induced ttT-2 cell proliferation when test compounds were carried over from the 1A5.7 cell cultures and were mixed with exogenous 1L-2. The lCso values shown have been corrected for the two-fold dilution of test compounds during this transfcr. NI = not inhibitory. Inhibition was less than 50% at nontoxic concentrations of test compound. d N E = not evaluable. High levels of toxicity and/or high levels of IL-2 inhibition preclude definitive evaluation of inhibitory activities of test compounds.

CS is well known to be a selective inhibitor of T-cell functions by blocking early stages of T-cell activation (Hess et al., 1981), including IL-l-induced T-cell activation (Palacios, 1981; Bendtzen and Petersen, 1982) and thymocyte proliferation (Gery et al., 1983). Our observations with IA5.7 cells are consistent with other T-cell-activation studies in which CS was shown to inhibit the production of IL-2, IL-3, interferon-}, and colonystimulating factor, but it did not block lL-2-induced T-cell proliferation (Hess et al., 1983; Herold et al., 1986; Kaufman et al., 1984). Inhibition of IL-2 production by CS in mitogen- and PMAstimulated human and murine T-cell lines was shown to be at the level of IL-2 m R N A transcription (Kr6nke et al., 1984; Elliott et al., 1984). The mechanism by which CS blocks T-cell lymphokine m R N A synthesis is unclear, but several studies point to various components of the plasma membrane or signal transduction mechanisms as targets. Among these are inhibition of lysolecithin acyltransferase, which plays a role in membrane phospholipid metabolism (Szamel et al., 1986); binding to a cytoplasmic protein called cyclophilin (Handschumacher et al., 1984), which may be the

same as calmodulin (Columbani et al., 1985), and the subsequent inhibition of intracellular Ca a + flux; or actual binding to the IL-1 receptor (Bendtzen and Dinarello, 1984) or the antigen receptor (Kaufmann et al., 1984). Recent evidence with several cyclosporine analogs suggests that calmodulin binding alone cannot account for the inhibitory effects of CS on T cells (Le Grue et al., 1986). Our observation that CS inhibitits IL-1/PHA-induced I1.-2 production during the early and late stages argues against CS being an IL-1-receptor antagonist, since such an agent should not inhibit the IL-2-production stage which occurs after exogenous IL-I is washed away. Differences in our studies from those of Bendtzen and Dinarello (1984), may reflect differences in the T cells used (they used human blood T cells) or in their use of a concentrations of CS five-hundred fold higher than the ICso on 1A5.7 cells. We are unaware of any previous studies of the effects of PGE2, PGF2~, cyclic nucleotides or theophylline on IL-l-dependent IL-2 production by T cells, but our findings are consistent with some studies of these agents in T-cell mitogenesis (Baker et al., 1981; Rappaport and Dodge, 1982; Tilden

61 and Balch, 1982; Maca, 1983). In particular, these studies have demonstrated that PGE2, but not PGF2~, inhibits IL-2 production and IL-2-induced human and murine T-cell proliferation with IC~o values in the range of 10 -6 to 10 8 M. Our findings the PGE2 inhibits these functions in murine 1A5.7 and HT-2 cells at somewhat higher concentrations ( I C 5 o ~ 5 - 8 × 10 6 M) may simply reflect different sensitivities of the murine cell lines and human lymphocytes to PGE2, as has been seen with other cell lines (Fadda et al., 1980). Our observations that theophylline, a drug which elevates intracellular levels of c A M P by inhibiting phosphodiesterases, and DBcAMP, but not D B c G M P , also inhibited IL-1/PHA-induced IL-2 production and IL-2-induced T-cell proliferation, suggest that a likely mechanism by which PGE2 may inhibit these activities is also via elevation of intracellular c A M P levels. The concentrations at which PGE2, theophylline and D B c A M P were active are similar to those known to elevate lymphocyte intracellular c A M P levels (Henney et al., 1972). PGF2~ does not elevate c A M P levels in lymphocytes. Our data suggest that the cAMP-active drugs PGE2, theophylline, and D B c A M P act only during the programming stage of IL-1/PHA-induced IL-2 production. Although the cAMP-active drugs, including PGE2, inhibited IL-1/PHA and IL-2 activities, PGE2 may additionally have acted though inhibition of protein phosphorylation, as suggested in several other studies (Chaplin et al., 1980; Maca, 1983). The high levels of D B c G M P needed for partial inhibition of IL-1/PHA activity during the programming stage (seen only at 10 -3 M) suggest that these effects may be nonspecific. The levels are at least 30-fold higher than the comparable effects of DBcGMP. In addition, it would be paradoxical if D B c G M P did inhibit IL-1/PHA activity, since both IL-1 and P H A have been shown to elevate intracellular c G M P levels in lymphocytes (Katz et al., 1978; Coffey et al., 1977). In summary, these studies show that HC, CS, and cAMP-active drugs inhibit IL-1/PHA-induced IL2 production in 1A5.7 cells. Each of these drugs clearly inhibits during the programming stage of activation and at least H C and CS also inhibit dur-

ing the IL-2-production stage. Although none of the agents tested could be shown to be IL-l-receptor antagonists in our bioassays, direct competitive-binding assays with radiolabeled IL-1 will be required for definitive evaluation of their IL-1 receptor-binding capabilities. As the mechanisms by which these drugs act are further clarified, these pharmacological studies will assist in understanding the biochemical mechanisms of IL-1 and IL-2 activation of T cells.

Acknowledgements We would like to thank Mary Elizabeth Bell and Mrs. Irena H r a b o w y for excellent secretarial assistance in the preparation of this manuscript.

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Kaufmann Y, Chang AE. Robb RJ, Rosenberg SA. Mechanism ol'action ofcyclosporin A: inhibition oflymphokinc secretion studied with antigen-stimulated T cell hybridomas..1 hmnu nol 1984:133:3107. Kaye. ,1, .laneway CA Jr. Induction of receptors for interlcukin-2 requires T cell Ag:la receptor crosslinking and interleukin-I. Lymphokine Res 1984:3:175. KrSnke M, Leonard WJ. Deppcr JM. Arya SK, Wong-Staal 17. Gallo RC, Waldmann TA, Greenc WC. Cyclosporin A inhints T cell growth factor gene expression at the level of mRNA transcription. Proc Nail Acad Sci USA 1984:81:5214. Larsson E-L. lscove NN, Coutinho A. Two distract factors are required for reduction o f t cell growth. Nature 198():283:664. LeGrue SJ, Turner R, Weisbrodt N, Dedman JR. Does the binding o[" cyclosporinc to cahnoldulin result in immunosupprcssion? Science 1986;234:68, Lowenthal JW, MacDonald H R. Binding and internalization of intcrleukin I by T cells. Direct evidence t\)r high and low aftinily classes of intcrleukin I receptor. J Exp Med 1986:164:1060. Maca RD. The effects of prostaglandins on the proliferation of cultured human T-lymphocytes. hnmunopharmalcology [983:6:267. Mizel S. lnterleukin-I and T cell activation. Immunol Rev 1982;63:51. Mossman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J hnmunol Methods 1983:65:55. Palacios R. HLA-DR antigens render interleukin-2-producer T-lymphocytes sensitive to interleukin-I. Scand J lmmunol 1981;14:321. Palacios R, Sugawara I. Hydrocortisone abrogates proliferation of T cells in autologous mixed lymphocyte reaction by rendering the interleukin-2 producer T cells unresponsive to interleukin-I and unable to synthesize the T cell growth factor. Scand J lmmunol 1982:15:25. Rappoport RS, Dodge GR. 1982; Prostaglandm E inhibits the production of human interleukm-2. J Exp Med 1982:155:943. Smith KA. Ruscetti FW. f cell growth factor and the cullure of cloned functional T cells. Adv lmmunol 1981:3 I:137. Szamel M, Berger P, Resch K. Inhibition of T-lymphocyte activation by eyclosporin A: interference with early activation of plasma membrane phospholipid metabolism. J lmmunol 1986;I 36:264. Tilden AB. Balch CM. Comparisml of PGE2 effects on human suppressor cell function and on interleukin-2 function. J hnmuno] 1982:129:2469. Zlotnick A, Daine B, Activation of l[.-l-dependent and IL-Iindependent T cell lines by calcium ionophore and phorbol esters. J lmmunol 1986:136:1033.