Clin. exp. Immunol. (1989) 76, 252-257

The effects of selected drugs, including chlorpromazine and non-steroidalanti-inflammatory agents, on polyclonal IgG synthesis and interleukin 1

production by human peripheral blood mononuclear cells in vitro

F. MARTINEZ & J. W. COLEMAN Department of Pharmacology and Therapeutics,University of Liverpool, Liverpool.

(Acceptedfor publication 8 March 1989)

SUMMARY

We tested a range of drugs for their effects on in vitro polyclonal IgG synthesis by human peripheralblood mononuclear cells (PBMC) stimulated with the lectin pokeweed mitogen (PWM). The testdrugs were selected on the basis of reported disruptive effects on immune function in vivo. IgGproduction between day 4 and days 7 or 8 of culture was measured by biotin-streptavidin sandwichELISA. The anti-psychotic agent chlorpromazine (0 55-1 7 tM) enhanced IgG synthesis to

approximately double control levels. In contrast, the non-steroidal anti-inflammatory drugs(NSAIDs) indomethacin, piroxicam, ibuprofen and aspirin inhibited IgG synthesis by up to 50%,with a rank order of potency that reflects their activity as inhibitors of cyclo-oxygenase. Phenytoin,procainamide, propylthiouracil, methimazole, D-penicillamine and D-penicillamine-L-cysteine allfailed to modulate IgG synthesis at non-toxic concentrations. The potentiation and inhibition of IgGsynthesis by chlorpromazine and indomethacin, respectively, was observed only when the drug waspresent during the first 24 h of culture. Neither chlorpromazine nor indomethacin, at non-toxicconcentrations, affected PHA- and PWM-stimulated proliferation ofPBMC. In addition, chlorpro-mazine, indomethacin and piroxicam, at concentrations which produced maximal modulation ofIgG synthesis, and D-penicillamine and D-penicillamine-L-cysteine at 10 pM failed to influenceproduction of interleukin- I-like activity. We conclude that chlorpromazine and NSAIDs, althoughthey exert opposite effects on IgG synthesis, act at an early stage ofB cell differentiation that appearsto be independent of interleukin 1 synthesis and early proliferative events.

Keywords IgG synthesis interleukin 1 immunoregulation chlorpromazine indomethacin

INTRODUCTION

The normal function ofimmunocompetent cells in vivo is subjectto modification by a wide range of drugs and/or their metabo-lites. Such disruption may lead to adverse reactions in whichantibody synthesis may either be depressed, leading to immunedeficiencies, or enhanced, leading to autoimmune disordersassociated with polyclonal B cell activation. For example, anti-nuclear antibodies have been detected in the serum of indi-viduals treated with chlorpromazine (Canoso & Sise, 1982),procainamide (Portanova et al., 1982; Rubin et al., 1986),phenytoin (Singsen, Fishman & Hanson, 1976) and propyl-thiouracil (Faber & Preben, 1966). D-penicillamine has beenshown to induce production of antinuclear antibodies in serumof some patients treated with the drug (Harkcom, Conn &Holley, 1978), and D-penicillamine-L-cysteine disulphide has

Correspondence: J. W. Coleman, Department of Pharmacologyand Therapeutics, University of Liverpool, P.O. Box 147, LiverpoolL69 3BX, UK.

been implicated as the active metabolite (Nakaike et al., 1983).Non-steroidal anti-inflammatory drugs, such as indomethacinand piroxicam, while not known to induce anti-nuclear anti-bodies, have been reported to influence humoral immuneresponses in humans in vivo (Goodwin et al., 1978; Goodwin,Ceuppens & Rodriguez, 1983).

In the present study we have investigated the effects of drugsreported to modulate immune function in vivo, but differingwidely in structure and pharmacological activity, for theireffects on human IgG synthesis in vitro. We also investigatedtheir effects on synthesis of the monocyte-derived growth factorinterleukin 1. The aim of the study was to develop an in-vitromodel of drug-induced disruption of antibody synthesis thatmight be of value for further investigation of the mechanismsinvolved in immunologically mediated adverse drug reactions.

MATERIALS AND METHODS

ReagentsThe following drugs were obtained from Sigma Chemical Co.Ltd., London, UK: piroxicam, ibuprofen, aspirin (acetylsalicy-

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lic acid), phenytoin (5,5-diphenylhydantoin), 6-n-propyl-2-thiouracil, methimazole, procainamide hydrochloride, chlor-promazine hydrochloride, D-penicillamine hydrochloride andpuromycin. D-penicillamine-L-cysteine disulphide and cyclos-porin A were generous gifts from Eli Lilly & Co. Ltd., Hants,UK and Sandoz Products Ltd., Middlesex, UK, respectively.Pokeweed mitogen (PWM) and concanavalin A (conA) wereobtained from Flow Laboratories Ltd., Herts, UK and phyto-haemagglutinin (PHA) from Wellcome Diagnostics, Dartford,UK. Recombinant human interleukin-lfI standard (rHuIL-If)was obtained from NIBSC, Herts, UK. Sterile microtitre plates(96-well, Nunclon) and RPMI-1640 medium were obtainedfrom Gibco Ltd., Uxbridge, UK. ELISA microtitre plates (96-well, Immulon B) were purchased from Dynatech LaboratoriesLtd., Surrey, UK. Affinity-purified rabbit anti-human IgG wasfrom Sera-Lab, Crawley, Sussex, UK, and horseradish peroxi-dase-streptavidin from Zymed Laboratories Inc., CambridgeBioscience, Cambridge, UK. The affinity-purified anti-humanIgG was biotin-labelled by incubation with 2-0 mg/ml of N-hydroxysuccinimidobiotin (Sigma) for 4 h at room temperature,followed by overnight dialysis against 1000 vol of 015 Mphosphate-buffered saline (PBS). 3H-Thymidine (22 Ci/mmol)was obtained from Amersham International, Bucks, UK.

Preparation ofperipheral blood mononuclear cellsHuman peripheral blood mononuclear cells (PBMC) wereobtained from healthy volunteers by centrifugation of hepari-nized blood over Lymphoprep (Nygaard & Co., Oslo, Norway)at 600g for 15 min. ThePBMC were washed four times in 0-15 Mphosphate-buffered saline (PBS, pH 7-2), once in RPMI-1640medium (300g, 10 min), and finally resuspended in RPMI-1640medium supplemented with 2-0 mM L-glutamine, 40 pg/mlgentamicin and 5% fetal calf serum (hereafter referred to ascomplete medium). Cell viability was assessed by dye exclusionfollowing incubation of the cells with 0 04% trypan blue for 5min at 37°C in complete medium, and was consistently found tobe > 97% at the start of cultures.

Effects ofdrugs on synthesis ofIgGPBMC (2-0 x 105 cells in 0-2 ml) were incubated in completemedium with a previously optimized concentration of PWM(0-5 pg/ml) and a range of concentrations of drug in sterilemicrotitre plates for 7-8 days at 37°C/5% CO2. At day 4 ofculture, 0-15 ml of supernatant medium was replaced with 0 15ml of fresh complete medium, and 3-4 days later supernatantfractions (0- 15 ml) were recovered following centrifugation (300g, 10 min), stored at - 20°C and subsequently assayed byELISA for total IgG content.

Sandwich ELISA for measurement of total IgGELISA microtitre plates were coated overnight at 4°C withaffinity-purified rabbit anti-human IgG (1:1000 in 0-05 Mcarbonate-bicarbonate buffer pH 9-6, 100 pl/well). The plateswere washed three times over a 2 min cycle in 0-15 M PBScontaining 0 05% Tween 20 (PBS-Tween), and shaken dry.Normal human serum, previously quantified for IgG contentagainst a commercially available standard serum preparation(Behring Institut, Marbug, FRG), was serially diluted three-fold (1:25 000-1:18 x 106, corresponding to 440-0-6 ng/ml ofIgG) in complete medium in the first column (seven wells) ofeach ELISA plate with complete medium only added to the

eighth well as a blank (100 p1/well). Culture supernatant testsamples were serially diluted three-fold (1:5-1:3645) in com-plete medium in the remaining columns (100 p1/well). Incuba-tion was for 1 h at room temperature. The plates were washedand biotin-labelled, affinity-purified anti-human IgG was addedfor 1 h (1: 1000 in PBS-Tween, 100 p1/well). The plates werewashed again, and streptavidin-peroxidase solution added for 1h (1:2500 in PBS-Tween, 100 p1/well). Finally, fresh substratesolution (containing 0-4 mg/ml o-phenylendiamine dihydro-chloride and 0-1% hydrogen peroxide in 0-15 M citrate-phosphate buffer, pH 5 0) was added (100 p1/well) to washedplates and the reaction stopped after 3 min with 50 p1 of 25%sulphuric acid. Optical density was determined at 490 nm on aDynatech MR600 plate reader interfaced with an Apple IImicrocomputer. The IgG content of test samples was deter-mined by measurement of the shift between parallel standardand test dilution curves.

Lymphocyte transformation assayHuman PBMC were obtained from venous blood as describedabove and incubated (2-0 x 105 cells in 0-2 ml) in the presence ofthe test drug and eitherPHA (5-0 pg/ml) orPWM (1-0 pg/ml) for72 h at 37°C/5% CO2. The cells were then pulsed with 3H-thymidine (0-5 pCi/well) and harvested 4 h later on to glass fibrefilters using a semi-automated cell harvester (Dynatech Mini-mash). Uptake of radioisotope was determined by liquidscintillation counting.

Assayfor interleukin-l-like activityInterleukin 1 (IL- I)-like activity was measured by the thymocyteco-stimulation assay. Briefly, thymocytes were isolated from 8-12 week-old male CBA mice and cultured in complete medium(3-0 x 105 cells in 0-2 ml) in the presence of serial dilutions ofrHuIL- 1# or test samples, and 1-0 pg/ml ofConA. Each samplewas assayed in triplicate. After 72 h of incubation (37°C/5%CO2), the cells were pulsed with 3H-thymidine (0 5 pCi/well) for18-20 h, and harvested on to glass fibre filters for determinationofcellular incorporation ofthe radioisotope. IL- I activity in testsamples was calculated as units/ml by measurement of the shiftbetween parallel standard and test dilution curves.

Effects of drugs on IL-I production by PBMCPBMC (106 cells/ml in 5 ml of complete medium) were culturedin the presence of 0-5 pg/ml PWM with or without the test drugin sterile plastic Petri dishes for 24 h at 37°C/5% C02. Cultureswere set up in duplicate and each experiment was performedthree times with PBMC from different donors. Followingincubation, supernatant fractions were removed, dialysedagainst 250 volumes ofRPMI-1640 medium containing 1% fetalcalf serum, then sterilized by filtration and assayed for IL-1activity as described above. Preliminary experiments had shownthat none of the test drugs, after dialysis, altered the responsive-ness of murine thymocytes to rHuIL-ll.

RESULTS

ELISA for total IgGELISA end-point activity (optical density) was sigmoidallyrelated to the concentration of IgG in standard serum (plottedlogarithmically) with mid-point activity at approximately 10 ng/ml (Fig. 1). Dilution curves obtained for test samples were

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(b)

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LI0-6 1-8 5-4 16 49 147 440 1/3645 1/1215

Standard (ng/ml) I

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Fig. 1. Typical standard (a) and test (b) dilution curves for IgGdetermined by biotin-streptavidin sandwich ELISA.

80 k

60 V

40 H

20 H

0 L L0.0XI 0.1 1.0 10

NSAID (#M)

Fig. 3. Effects on PWM-stimulated IgG synthesis by PBMC of theNSAIDs indomethacin (0); piroxicam (0); ibuprofen (0); aspirin (U).Results are means + s.e.m. for three experiments, each performed inquintuplicate.

_ I

0-19 0-55 1-7 5-0Chlorpromazine (,uM)

Fig. 2. Effect ofchlorpromazine on IgG synthesis (day 4 to day 7 or 8) byPBMC stimulated with PWM. Each plot represents a separate experi-ment (different blood donor). IgG synthesis is expressed as percentage ofcontrol release in the absence of drug (means + s.e.m. of quintuplicatecultures).

consistently parallel to dilution curves for IgG standards, andexhibited the same maxima (Fig. 1). The IgG content ofsupernatant fractions from PWM-stimulated PBMC variedbetween experiments from 0-1-2 Jg/ml.

Kinetics ofIgG synthesisPreliminary experiments showed that maximal IgG synthesis byPBMC occurred between day 4 and day 7 in PWM-stimulated

cultures. No increase in IgG synthesis was observed after day 8.Further experiments, in which PBMC were cultured in thepresence or absence of puromycin (an inhibitor of proteinsynthesis), demonstrated that > 90% of IgG released intosupernatant fractions between day 4 and day 7 or 8 was theproduct of active synthesis, rather than passively shed material.In contrast, during the first 4 days of culture, only 16% ofreleased IgG was actively synthesized (inhibited by puromycin).Consequently, in all subsequent experiments reported here,active synthesis was determined by measurement of IgGreleased between day 4 to day 7 or 8 of culture.

Effects of drugs on IgG synthesisChlorpromazine, within the concentration range 019-1b7 gIM,potentiated PWM-stimulated synthesis of IgG, with maximumenhancement (approximately a doubling ofresponse) seen at 1-7gm (Fig. 2). At a concentration of 5-0 JM, some loss of viablecells (approximately 20%) was observed; whereas at 15 gMcomplete toxicity was observed. These toxic effects may havecontributed to the fall in IgG synthesis at 5 JUM chlorpromazine.

In contrast to chlorpromazine, the four non-steroidal anti-inflammatory drugs (NSAIDs) tested all induced a dose-dependent inhibition of IgG synthesis (Fig. 3). The potency ofinhibition decreased in the order: indomethacin > piroxicam >

ibuprofen> aspirin, with IC25 values (concentrations giving25% inhibition) calculated from the data in Fig. 3 as 0-017, 0-17,0-84 and 5 7 AM, respectively. No loss of cell viability was

observed at inhibitory concentrations of NSAIDs.

254

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Effects of drugs on IgG synthesis

300

250 _

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Fig. 4. Effect of time of addition of chlorpromazine (2-0 aM, 0) andindomethacin (1 0 pM, 0) on IgG synthesis by PWM-stimulated PBMC.Results are mean ± s.e.m. for three experiments.

Of the other drugs tested, phenytoin, procainamide, propyl-thiouracil and methimazole exerted no effects on IgG synthesisat concentrations up to 100 MM. D-penicillamine and D-penicilla-mine-L-cysteine disulphide exerted no effects at concentrationsup to 10 UM, although at 100 gM both drugs induced inhibitionwhich was accompanied by loss of cell viability.

Time-dependence of the effects of chlorpromazine and indo-methacin on IgG synthesisThe potentiating effect ofchlorpromazine (2-0 pM) was maximalwhen the drug was added at the start of culture; the effect wasmarkedly reduced ifthe drug was added 24 h after the start oftheculture, and no effect was observed when the drug was added 48h after the start of culture (Fig. 4). The inhibitory effect ofindomethacin (IO- gM) was observed only when the drug was

added at the start of culture; addition after 24 h and at latertimes produced no effect (Fig. 4).

Effects of chlorpromazine and indomethacin on lymphocytetransformation in response to PHA and PWMChlorpromazine, at non-toxic concentrations within the range0-06-1-7 pm, exerted no significant effects on PHA and PWM-induced proliferation but at 5 giM inhibited proliferation to bothlectins (Table 1). Indomethacin, at 0-001-10 pm, exerted no

effects on lectin-induced proliferation ofPBMC (Table 1).

Effects ofdrugs on the production ofinterleukin-J-like activity byPBMCPWM-stimulated PBMC produced a five-fold increase in releaseof IL-l-like activity over unstimulated cells. Chlorpromazine,indomethacin and piroxicam did not affect PWM-stimulatedIL-I synthesis at the concentrations shown to modify synthesisof IgG. D-penicillamine and D-penicillamine-L-cysteine disul-phide (10 gM) were likewise without effect (Table 2).

Table 1. Effects of chlorpromazine and indomethacinon lymphocyte proliferation in response to PHA and

PWM

Drug concentration(PM) PHA (5 pg/ml) PWM (1 g/ml)

Chlorpromazine5-0 21-3+4-6* 35.0+12 1*1-7 799+ 14-3 86-6+28-60-55 92-0+4-7 98 7+8 50.19 86-1+7-1 993±770-06 85 6+6-9 97-6+ 5 00 100 100

Indomethacin100 114-7+15-0 1061+6-61.0 116-0+13-0 103 7+10 501 113-0+159 1055+1090 01 100-6+6-3 104 4+3-80 001 101-2+3-1 96 5+1-20 100 100

Values represent mean+ s.e.m. for three experi-ments (each performed in quintuplicate) and areexpressed as percentage of control 3H-thymidineuptake (ct/min) in the absence of drug. * P< 0 01 bypaired Student's t-test of non-normalized data.

Table 2. Effects of selected drugs on release of interleukin- 1-like activityby PWM-stimulated human PBMC in vitro

Release ofIL-l-like

Concentration activityDrug of drug (pM) (% control)

Chlorpromazine 2-0 104-8 + 19 5 (4)Indomethacin 0.1 98-7+19-1 (3)Piroxicam 1.0 85 3+ 13 9 (4)D-penicillamine 10 0 957 + 11-6 (3)D-penicillamine-L-cysteine 10 0 79-0+20-2 (3)

disulphide

Values shown are expressed as percentage of control release of IL- 1-like activity (units/ml) by PWM-stimulated cells in the absence of drug,and they represent mean + s.e.m. for the number of experimentsindicated in parentheses. IL-1-like activity released by unstimulatedPBMC in the absence of drug was 21-4+3-2% (n=4) of stimulatedcontrols. For all experiments, P > 0-05 (paired Student's t-test).

DISCUSSION

We report the results of studies in which a range of drugs weretested for modulatory effects on IgG synthesis by humanPBMCstimulated with the polyclonal activator PWM.

Chlorpromazine was found to enhance human IgG synthesisin vitro, an observation which accords with in vivo studiesshowing elevations in serum IgM (Zarrabi et al., 1979) and anti-

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256 F. Martinez & J. W. Coleman

nuclear antibodies (Canoso & Sise, 1982) in patients treatedwith the drug. Ichimura (1978) found that plaque-forming cellresponses by murine spleen cells were enhanced slightly by lowconcentrations of chlorpromazine (0 5-5 jM), which is againconsistent with a potentiating effect of the drug on antibodysynthesis. In the present study the concentration ofchlorproma-zine inducing maximal enhancement of IgG synthesis (1-2 gM)was within the range ofplasma levels found clinically (0 028-2-8gm; Curry, 1970). Chlorpromazine enhanced IgG synthesis onlywhen present during the first 24 h of culture, i.e. at a stagepreceding active IgG synthesis, but exerted no effect at non-toxic concentrations on synthesis of IL-1-like activity or onproliferation induced by the T cell mitogen, PHA, and the T+ Bcell mitogen, PWM. Since we found no effects on IL- I synthesisand, since generation of interleukin 2 is an integral event inPWM-stimulated mitogenesis (Nakagawa et al., 1987), weconclude that chlorpromazine does not enhance IgG synthesisby modulating the release of these two lymphokines but, moreprobably, acts on other regulatory mechanisms involved inactivation ofB cells. Previous reports (Nahas, Desoize & Leger,1979; Barbu et al., 1987) have shown inhibitory effects ofchlorpromazine on mitogen-induced human lymphocyte pro-liferation, but at concentrations of the drug higher (a 5 gM)than those that we found to enhance IgG synthesis and which,from our own observations, we suspect to be toxic.

Chlorpromazine possesses a number of biochemical andpharmacological activities. For example, in common with otherphenothiazines, it inhibits calmodulin. It also acts as anantagonist at 13-adrenergic, dopamine, histamine and 5-hydroxytryptamine receptors. The presence of fl-adrenergic,histamine and 5-hydroxytryptamine receptors on lymphocyteshas been demonstrated (Kedar & Bonavida, 1974; Bishopric,Cohen & Lefkowitz, 1980; Bonnet, Lespinats & Burtin, 1987).There is probably no endogenous /3-adrenergic agonist inPBMC cultures, although histamine, which is now known toderive from macrophages (Oh et al., 1988) as well as mast cellsand basophils, and 5-hydroxytryptamine (platelet-derived) maybe present at low concentrations. Experiments are in progress toassess whether calmodulin, or histamine and/or 5-hydroxytryp-tamine receptors may be the target for chlorpromazine-inducedenhancement of IgG synthesis.

The concentrations of indomethacin used in these experi-ments are within the range ofplasma drug levels attained in vivo(for example, following an oral dose of 100 mg indomethacin,plasma levels reached 18 gm; Emori et al., 1976). In addition, therank order of potency of NSAIDs (indomethacin > piroxicam> ibuprofen > aspirin) that we showed to inhibit IgG synthesis,and their IC25 values, are consistent with the effect being viainhibition of cyclo-oxygenase (Flower, 1974; Brune et al., 1981;Neuman et al., 1987). Ceuppins & Goodwin (1982) reportedthat PGE2 is produced endogenously inPBMC cultures and actsto augment immunoglobulin synthesis. In agreement with thepresent study they also showed that NSAIDs inhibit IgGsynthesis by up to 50%. Thus it is evident that prostaglandins donot provide an obligatory signal for antibody synthesis, butrather act to potentiate synthesis to approximately twice thatobtained in the absence of prostaglandins. Ceuppins & Good-win (1982) obtained evidence that the action of PGE2 was via aradiosensitive CD8-positive T cell which they postulated wasresponsible for suppression of IgG synthesis.

Indomethacin, like chlorpromazine, modulated IgG synthe-

sis only when present during the first day of culture, but had noeffect on synthesis of IL-I or proliferation ofPBMC stimulatedwith PHA and PWM. These results suggest that NSAIDs alsoact, presumably by controlling PGE2 levels, at an early stage ofB cell differentiation which is independent of the release ofinterleukins I and 2. Reports that proliferation of PHA-stimulated PBMC is inhibited by PGEI and PGE2 (Goodwin,Bankhurst & Messner, 1977) and enhanced by indomethacin(Goodwin, Messner& Peake, 1978; Williams & Davidson, 1983)are difficult to reconcile with our own results and do not helpexplain the inhibitory effects of NSAIDs on IgG synthesisreported here and by Ceuppins & Goodwin (1982) unless onesuggests again a selective effect of prostaglandins on suppressorT cells. The reported potentiation of IL-I synthesis by NSAIDsin vitro (Sandborg et al., 1985; Repo et al., 1988) was notobserved in the present study, or in that of Otterness et al.(1988). Otterness et al. (1988) showed that the action ofNSAIDsis to abrogate inhibition of the thymocyte IL-1 assay byprostaglandins, rather than to inhibit IL-l release. In addition,we have found that, because of the non-linear relationshipbetween IL- I activity and thymocyte proliferation, it is essentialto measure displacement of parallel test and standard dilutioncurves to obtain accurate measurements of activity.

Finally, the lack of effect of phenytoin, procainamide,propylthiouracil, methimazole, D-penicillamine and D-penicilla-mine-L-cysteine in the present study suggests that their actionson human immune responses (see introduction) do not extend tomodulation of polyclonal IgG synthesis in vitro. Although thegrowth and function ofhuman cells in vitro is not subject to all invivo regulatory mechanisms, cell culture systems allow the studyof drug effects on cells in a manner unimpeded by suchregulatory processes, and hence elucidation of mechanismswhich may result ultimately in immune dysfunction in vivo.Thus, the unexpected finding that chlorpromazine elicits anaugmentative effect on polyclonal IgG synthesis in vitro mayrepresent an important aspect of the mechanism by which thisdrug induces the formation of anti-nuclear antibody in vivo, andhence contributes to the pathogenesis of drug-induced auto-immune disease.

ACKNOWLEDGMENTSF.M. is supported by an MRC studentship. We thank Miss SusanOliphant for typing the manuscript.

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