inhibition of antigen- mitogen-induced lymphocyte proliferation … · adherent cells; pbm,...

Inhibition of Antigen- and Mitogen-Induced Human

Lymphocyte Proliferation by Gold Compounds

PETERE. LIPSKY and MORRISZIFF

Froml the Rhleuminatic Diseases Unit, Department of Internal Medicine, The University of TexasSoiitlhtwestern Mlledical School, Dallas, Texas 75235

A B S T RA C T Gold sodium thiomalate (GST) in-hibited in vitro antigeni- and mitogen-triggered htlmanilymphocyte DNAsynthesis. Inhibition of responsive-ness was observed with concentrations of GSTequiva-lent to gold levels fouind in serunm or tissues of pa-tients receiving chrysotherapy. Inhibition was de-pendent upon the gold ion itself since GSTand goldchloride were both inhibitory whereas thiomalicacid was not. Inhibition couild not be explained bynonspecific killing of cells or by an alteration in thekinetics of the responses. GST inhibited mitogen-induced proliferation most effectively when presentfrom the initiation of culture and could not inhibitthe responsiveness of cells which previously hadbeeni activated by concanavalin A. These findingsindicated that GST blocked a critical early step inlymphocyte activation. The degree of GST-indtucedinhibition of proliferation was increased in ctultturesof cells partially depleted of monocytes. Moreover,inhibition was reversed by supplementation of thesectulttures witlh purified monocytes. These observationssuggested that GST blocked thymus-derived (T)-lymphocyte activationi by interfering with a requiisitefunction of the monocyte poptulation in initiating stuchresponses. Prolonged incubation of peripheral bloodmononuclear cells with GST resulted in diminishedmitogen responsiveness uipon subsequient cultturein the absence of gold. The addition of fresh mono-cytes restored responsiveness to these populations.Fturthermore, preinctubation of pturified monocyteswith GST rendered them deficient in their ability tostupport mitogen-induced T-lymphocyte proliferation

This work xwas presented in part at the Anntual ScientificMeeting of the Americani Rheuimatism Association, Chicago,Ill., 11 juine 1976.

Dr. Lipsky is a recipient of a Postdoctoral Fellowshipfrom the Arthritis Foundation. Dr. Ziff is a recipient of aU. S. Public Health Service Research Career Award.

Received for publication 30 Atuguist 1976 and in revisedform 15 Noviember 1976.

on subsequent cultture. These observations indicatethat the major effect of GST restults from interferencewith the ftunctional capability of the monocyte poptula-tion.

INTRODUCTION

Rheuimatoid arthritis is a disease characterized bychronic inflammnation of synovial tisstues. Although itsetiology remainis unknown, immunnological processeshave been implicated in the pathogenesis and main-tenance of the chronic synovitis (1). Thus, cells ofbone marrow origin, along with their secretory prod-ucts, play a role in this process as indicated by thepresence, within rheulmatoid synovial tissue, of cellswhiclh syntlhesize and secrete both immuinoglobtulinand rheumlnatoid factor locally (2-4). More recently, arole for thymuis-derived (T)l lymphocytes also hasbeen stuggested by the demonstration of large num-bers of T lymphocytes as compared to bone-marrowderived lymphocytes (B lymphocytes), both in synovialfluid (5-7) and digests of synovial tissue (8, 9).

Chrysotherapy has been tused in the treatment ofrheumatoid arthritis for nearly 50 years. Recent clinicaltrials have established that treatment with gold com-pounds not only can ameliorate the symptoms ofrheumatoid arthritis, buit also can slow or even haltthe progress of the disease (10-13). These observa-tions imply that gold compouinds exert a potent anti-inflammatory effect in vivo. Since immune mechanisms

IAbbreviation.s used in this p)ap)er: B lymphocyte (or Bcell), bone-marrow derived lymphocyte; Con A, concanavalinA; Aecpm, difference in counts per minute between the meansoftriplicate-stimtilated and control ctultures; FBS, fetal bovineserum; GST, gold sodium thiomalate; 2-ME, 2-mercapto-ethanol; MLR, mixed leukocyte reaction; M'), monocyte ormacrophage; PBL, peripheral blood lymphocyte depleted ofadherent cells; PBM, peripheral blood mononuclear cell;PHA, phytohemagglutinin; PWM, pokeweed mitogen;SK/SD, streptokinase-streptodomase; T lymphocyte (or Tcell), thymtis-derived lymphocyte; TMA, thiomalic acid.

The Jou rnal of Clitnical In vestigationi Volti me .59 March 1977 4. 5 -466 455

are thought to mediate the chronic inflammatorysynovitis of rheumatoid arthritis, it seems reasonableto suggest that the therapeutic efficacy of gold com-pounds may relate to their ability to modify immuno-logical processes.

The studies reported here were undertaken, there-fore, to delineate the effect of gold compounds oncertain in vitro correlates of immune responsiveness.Specifically, the action of gold sodium thiomalate(GST) has been investigated, with respect to itsability to modify in vitro antigen- and mitogen-induced proliferative responses in populations ofnormal human peripheral blood mononuclear cells.These proliferative responses share two characteristics.First, the primary responding cells are T lymphocytes(14-16), and, second, the initiation of each of theseresponses is dependent upon the active participationof a nonresponding accessory cell (17-22). Inpopulations of human peripheral blood mononuclearcells, the monocyte subserves this ftunction (15, 18,19, 21, 22).

GSTinhibited antigen- and mitogen-induced humanlymphocyte proliferation. Inhibition, which wasmediated by the gold moiety itself and not the sulfur-containing ligand, was concentration dependent anddemonstrable within the range of serum or tissue levelsfound in patients treated with gold compounds. Twomajor effects of GST were observed. The first was areversible interference with lymphocyte activationwhich required the presence of the gold compound inthe culture medium. The second was an irreversiblediminution in the magnitude of responsiveness whichdeveloped after prolonged exposure to GST, but nolonger required the presence of the gold compoundin the culture to be manifest. Data are presented toindicate that neither effect resulted from an action ofGST on the potential responsiveness of the lympho-cyte population per se. Rather, in both cases, GSTinterfered with the ability of monocytes to serve aseffective accessory cells in the initiation of theseproliferative responses.

METHODSCell preparation. Venous blood was obtained from

healthy young adults who were taking no medications atthe time of study. The mononuclear cell population wasobtained from heparinized whole blood by centrifugationon sodium metrizoate/Ficoll cushions (Lymphoprep, Nye-gaard & Co., Oslo, Norway) as previously described (23).Resultant preparations of peripheral blood mononuclearcells (PBM) consisted of 70-85% lymphocytes and 15-30%monocytes.

Mitogens and antigens. The following stimuli were used:concanavalin A (Con A, ICN Nutritional Biochemicals Div.,International Chemical & Nuclear Corp. Cleveland, Ohio),purified phytohemagglutinin (PHA, The Wellcome Re-search Laboratories, Beckenham, England), pokeweed mito-gen (PWM, Grand Island Biological Co., Grand Island,

N. Y.), streptokinase-streptodomase (SK/SD, Lederle Labora-tories, Pearl River, N. Y.).

Gold compounds. The following compounds wereutilized: gold sodium thiomalate (GST, Merck ChemicalDiv., Merck, Sharp & Dohme & Co., Inc., Rahway, N. J.),Gold trichloride (AuCl3 * HCI * 3H20, J. T. Baker ChemicalCo., Phillipsburg, N. J.), and d, l-thiomalic acid (TMA,Merck, Sharp & Dohme & Co., Inc.).

Miscellaneous reagents. The following reagents wereused where indicated: 2-mercaptoethanol (2-ME, Eastman-Kodak Co., Rochester, N. Y.) and a-methyl-D-mannoside andmitomycin-C (Sigma Chemical Co., St. Louis, Mo.).

Culture medium. Cultures were carried out in mediumRPMI-1640 (Microbiological Associates, Bethesda, Md.)supplemented with 0.3 mg/ml fresh l-glutamine, 10 ug/mlgentamicin, and 200 U/ml penicillin-G. The medium wasfurther supplemented with either 10% fetal bovine serum(FBS, Microbiological Associates) or 10% heat-inactivated,pooled human AB serum.

Separation of PBM into adherent and nonadherentpopulations. In certain experiments, PBMwere separatedinto adherent and nonadherent populations. This was ac-complished by suspending PBM (5 x 106/ml) in mediumRPMI supplemented with 10% FBS and incubating themin 100 x 20-mm glass Petri dishes for 60 min at 37°C.After this incubation, the nonadherent cells were removedby washing with warm medium and then pooled. The cellsadherent to the glass Petri dish were then reincubated inmedium containing 10% FBS for 30 min at 37°C afterwhich they were vigorously washed with four changes ofwarm medium to remove loosely adherent cells. The ad-herent cells remaining after these washings were thenbathed in phosphate-buffered saline (pH 7.2), chilled to4°C, and dislodged from the Petri dish with a rubber police-man. After washing, the cells were suspended in medium,exposed to 40 ,Ag/ml mitomycin-C for 60 min at 37°C, andwashed four times. More than 90% of the resulting cellpopulation was monocyte/macrophages as judged eithermorphologically with Euchrysine staining (24) or bydetermination of phagocytic cells using polystyrene latexparticles. Viability was greater than 95% as estimated bytrypan blue exclusion.

The nonadherent cells removed from the Petri dish on thefirst wash were pooled and further depleted of adherentcells by passage over nylon-wool (Fenwal Laboratories,Inc., Morton Grove, Ill.) columns, as previously described(20). The resultant population contained about 85-90%T lymphocytes as judged by rosette formation with sheeperythrocytes (25), and 0.1-0.8% monocytes as detectedeither morphologically with Euchrysine staining or bydetermination of phagocytically active cells.

Technique of cell culture and assay of lymphocyte DNAsynthesis. Cultures were carried out in sterile microtiterplates with round-bottomed wells (Cooke Engineering Co.,Alexandria, Va.). In some experiments, as noted in the text,cells were cultured in sterile microtiter plates with flat-bottomed wells (Falcon Plastics, Division of BioQuest, Ox-nard, Calif.). Routine cultures were done in triplicate, witheach well containing 1 x 105 responding cells in 0.2 ml of cul-ture medium. In some experiments, the number of cellscultured per well was varied; in others, the respondingcells were supplemented with mitomycin-C-treated mono-cyte/macrophages. The stimulating agent or an equal volumeof medium as control was added directly to the cultures atthe indicated final concentrations. Two-way mixed leukocytereactions (MLR) were set up by mixing 5 x 104 PBMfromone individual with 5 x 104 PBM from another unrelatedindividual in triplicate 0.2-ml cultures. Mitogen-stimulated

456 P. E. Lipsky and M. Ziff

TABLE IGSTInhibition of HumanPBMProliferation

Lymphocyte DNAsynthesis*GSTtconen. PHA Con A SK/SD MLRpg/ml (n = 15) (n = 19) (n = 4) (n =4)

Acpmt

0 85,105+6,013 56,648+3,857 26,844+2,953 29,685+9,298

%Inhibition"

1 4.3+2.8 5.6+2.7 13.7+5.3 0.8+2.910 15.9+1.5 20.0+2.2 27.0±4.3 21.1+4.825 21.5±1.9 23.8±1.9 42.5±6.0 47.6±+11.750 26.1± 1.7 34.4±2.6 75.2±3.8 89.7±3.8

100 35.6±3.9 6.1.3±2.9 96.9±2.9 99.4±0.4

* [3H]Thymidine incorporation observed in cultures containing 1 x 105 respondingcells per round-bottomed well using previously determined optimal concentrationsof stimuli (PHA = 0.5 ,g/ml, Con A = 5.0 ,g/ml, SK/SD = 50,ug/ml). Mitogen-stim-ulated cultures were incubated for 3 days, antigen-stimulated cultures for 5 days,and MLRcultures for 7 days. Data are expressed as mean±SEMfrom n separateexperiments.t Refers to concentration of GSTof which gold constituted 50.5%.§ Background [3H]thymidine incorporation of unstimulated cultures from 19 sep-arate experiments, Acpm = 1,252±180. Background Acpm in cultures containingGST: 1 ,ug/ml, 1,343±249; 10 ,ug/ml, 1,320±224; 25 ,ug/ml, 1,248+259; 50 ,ug/ml,1,192+254; 100,ug/ml, 809±169."Inhibition by 1 ,.ug/ml GSTnot significant. Each response is significantly inhibitedby all other concentrations of GST (10-100 ,ug/ml). Inhibition is significant atP < 0.001 (paired sample t test) for each situation except for inhibition of SK/SDresponse by 10 jig/ml GST (P < 0.05) and 25 ug/ml (P < 0.01), and inhibition ofMLRby 10 and 25 ,ug/ml GST (P < 0.02).

cultures were carried out in medium supplemented with 10%FBS whereas antigen-stimulated and MLR cultureswere supplemented with 10% human serum. Cultures wereincubated (72 h for mitogen stimulation, 120 h for antigenstimulation, and 168 h for MLR) at 37°C in a humidifiedatmosphere of 5% CO2 and 95% air. 18 h before harvesting,1 ,uCi of tritiated thymidine (6.7 Ci/mM, New EnglandNuclear, Boston, Mass.) was added to each well. Cellswere harvested onto glass fiber filter paper using a semi-automated microharvesting device. Tritiated thymidineincorporation was then determined by liquid scintillationspectroscopy. All data are expressed as the difference incounts per minute between the means of triplicate stimulatedand control cultures (Aepm). In some experiments, data areexpressed as percent inhibition compared to control stimula-tion calculated according to the following formula: %inhibition = (1-[Acpmi/Acpml]) x 100, where Acpmi = pro-liferative response in the presence of a putative inhibitorand Acpm, = proliferative response in control cultures.

RESULTS

GSTinhibition of human lymphocyte proliferation.GST inhibited the proliferative responses of normalhuman lymphocytes induced by either of the mitogensPHA or Con A, the antigen SK/SD or allogeniclymphocytes in a MLRas depicted in Table I. The

degree of suppression observed was dependent on theconcentration of GST, with significant inhibition seenwith 10 ug/ml. The data shown in Table I were ob-tained using concentrations of mitogens or antigenswhich gave maximum responses in control culturescontaining no gold. When suboptimal concentrationsof mitogen were employed, the degree of GSTinhibi-tion of lymphocyte proliferation was markedly greaterthan that observed in cultures stimulated with con-centrations yielding maximal [3H]thymidine incorpora-tion (Table II). Moreover, the concentration or natureof the serum used to support the cultures appeared tohave little effect on GST inhibition. Thus, varyingthe concentration of FBS from 5 to 20% had no signifi-cant effect on GST inhibition of the PHA response.Furthermore, GSTsuppression of PHAresponsivenesswas similar in cultures supported by either humanserum or FBS (data not shown).

Experiments were carried out to determine whetherGST caused an actual depression in the degree ofmitogen-induced lymphocyte proliferation or merelyaltered the kinetics of these responses (Fig. 1). Inthese studies, PBMwere cultured for 2-6 days with

Gold Inhibition of Lymphocyte DNASynthesis 457

TABLE IIGST Inhibition of Optimal and Suboptimal

Mitogenic Stimulation

Lymphocyte DNAsynthesis*

cs-Ft Con A PHA8S1Lg/mI jtg/mi 5 jug,mnl 0.1 g/m11 0.5 gog/mI

AcpIn

0 15,315 49,800 51,198 63,220

% Iftliibitiorl

10 48.9 19.5 28.4 13.925 68.8 42.1 50.5 34.8

* [3H]Thymidine incorporation observed in cultures con-taining 1 x 105 responding cells per round-bottomed well.Each term represents the mean of triplicate determinations.

PHA, Con A, or medium control in the presence orabsence of 10 ,ug/ml GST, and [3H ]thymidine incorpora-tion was assayed daily. Lymphocyte DNAsynthesis in-duced by PHA and Con A in the absence of GST isshown in Fig. 1 (insert) as a functioni of the length ofculture. Peak response to both mitogens is seen aftera 3-day incubation. GST-mediated suppression of eachof these responses was observed throughout the periodof observation. There was no tendency for the degreeof lymphocyte proliferation in GST-inhlibited cul-tures to approachi that manifested by conitrol cultures.On the contrarv, as inculbation wvas proloniged beyond3 days, the degree of GST-mediated inhibition be-came more marked, suggesting the possibility that pro-longed incubation in GST may have an adverseeffect on lymphocyte responsiveness. This point willbe addressed in more detail in a subsequent section.For the remaining experiments, a standard 3-day in-cubation was employed to analyze the effect of GSTonmitogen stimulation.

To determine whether suppression of lymphocyteproliferation re(quired the intact gold thio moleculeor merely the sulfuir-containing ligand, the inhibitoryeffect of GST was compared to that of thiomalicacid (TMA). Fig. 2 depicts the percent inhibition ofthe Con A response as a function of the molar con-centration of each of these reagents. TMAcaused nosignificant inhibition of the Con A response and GSTproduced significant suppression at concentrations aslow as 26 ,uM (10 ,ug/ml).

The effect of gold chloride (AuCl3 HCI 3H20)on Con A responsiveness was next examined to ascer-tain whether the inhibitory capacity of gold com-pounds was limited to those in whiich gold was com-plexed to a sulfur-containing ligand (Table III). Theability of gold chloride to inhibit Con A-inducedlymphocyte proliferation was similar to that manifested


by GST at a low concentration (10 ,ug/ml) buit ex-ceeded the inhibitory capacity of GST at higher con-centrations (25, 50 ,ug/mll). These data suggest thatinhibition of lymphocyte proliferation indtuced bygold compotunds is mediated by the gold moietyitself and is not depenident on the ligand.

Effect of immiiiiediate an,d delayed additioni o( GSTon lymnphocyIte proliferationi indtuced byi Cott A.Lymphocyte DNAsynithesis assayed by [3H ]thvmllidineincorporationi is a relatively late ocecurrence in a com-plex series of biological events triggered by imiitogens(26, 27). To determine whether there was a particularstep in this sequence of events which was sensitiveto inhibition by gold, GST was added to control andCon A-stiiitlated culttures either at the initiation of theinccubation or at varying times thereafter (Fig. 3).The total length of inctubation was 72 h for each ex-perimental point. The stippled area indicates the ConA response of these cells (mean Acpm+SEM) whenincubated for 72 h witlh no added gold. Wileni GSTwas added to the cultures at their inceptioni, conceni-

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FIGURE 1 GST-mediated inhibition of the response of PBMto Con A and PHAas a function of the length of incubation.PBM(1 x 105/well) were incubated with 5 Ag/ml Con A, 0.5,ug/ml PHA, or an equal volume of medium as control for2-6 days at 37°C in the presence or absence of 10 ,ug/mlGST and assayed for [3H]thymidine incorporation. Insertdepicts proliferative response (Acpm) of control cells cul-tured without gold. Each point represents the mean±SEMof three separate experiments.

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tration-dependent inhibition of the Con A responsewas observed with 25 ,ug/ml resulting in a 30.8%inhibition, and 100 ,tg/ml suppressing the responseby 68.5%. When the addition of GST was delayedby progressively longer intervals from the beginning ofthe culture, as indicated on the abscissa, the degreeof resultant inhibition became progressively lessmarked. There was no effect on proliferation whenGSTwas present only for the terminal 24 h of culture.These findings indicate that GST inhibition does notresult merely from a nonspecific blocking of eitherthymidine uptake or DNAsynthesis by lymphocytes.Moreover, the observation that inhibition by gold wasmost effective when GST was present from theinitiation of culture suggests that gold acts by blockingan early step in the sequence of events resulting inlymphocyte DNAsynthesis. Similar results were ob-served when PHAwas used as the stimulus or whengold chloride was used as the inhibitor (data notshown).

Inability of GST to inihibit DNA sytnthesis by ConA-activuated lymphocytes. The previous experi-ments suggested that the major effect of gold was toinhibit an early step in the sequence of mitogen-triggered events resulting in lymphocyte DNA syn-thesis. To investigate this possibility further, theeffect of GST on the degree of lymphocyte prolifera-tion, manifested by a population of PBMpreviouslyactivated by Con A in the absence of gold, was in-vestigated (Table IV). PBM were incubated with 5,g/ml Con A or an equal volume of medium as con-trol for 24 h at 37°C. At the end of this period of activa-tion, the cells were incubated twice with 40 mMa-methyl-D-mannoside for 10 min at room temperatureand washed to remove excess Con A (28). The cells

TABLE IIIGold Inhibition of ConA-Induced Lymphocyte Proliferation:

Comparison of GSTand Gold Chloride

[3H]Thymidine incorporationConcenitration

of inhibitor GST* Gold chloridet

pg/mi1 % Inhibition §

10 17.0+3.2 14.5+4.525 24.8+4.7 52.0+8.350 42.3±6.6 91.4+3.3

* GST; gold content: 50.5%.t Gold Trichlonde (AuCl3 HCl 3H20); gold content: 49.2%.§ Each term represents the mean+SEMof five separate ex-periments.

then were suspended in fresh medium, supplementedwith 10% FBS, and aliquoted into the wells of micro-titer plates (1 x 105/well). If the cells were assayedfor [3H]thymidine incorporation at this point, nosignificant difference was noted between Con A-activated and control cells (data not shown). How-ever, when the cells were incubated at 37°C for 48 hafter the period of mitogen exposure, significant[3H]thymidine incorporation developed in the ConA-activated population (ACPM = 22,179t 1,545). As

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FIGURE 3 Effect of delayed addition of GST on Con A-induced lymphocyte DNAsynthesis. PBMwere cultured for72 h with or without 5 ,Lg/ml Con A. The proliferative re-sponse of these cells cultured without GST is indicated bythe stippled area (Acpm = 71,924+4,036). 25 or 100 ,ug/mlGSTwas added either at the initiation of culture or at varyingtimes thereafter. Each point represents the mean of triplicatedeterminations.

Gold Inhibition of Lymphocyte DNASyntthesis 459

TABLE IVFailure of GST to Inhibit DNASynthesis of Lymphocytes

Previously Activated by Con A

DNAsynthesis by Con A-activated PBM*GSTconcn. dtiring culttured with:

last 48 h ofincubation Medium Con A

pglmI [3H ] Thymidine incorporation-Acpin I

0 22,179±1,545 95,995+2,58910 20,952±1,421 82,626±3,05825 22,162±2,643 76,637±2,98850 21,155±2,523 65,525±1,352

* PBMwere incubated in test tubes (1 x 106/ml in mediumcontaining 10% FBS) with 5 ,ug/ml Con A or an equal vol-ume of medium as control for 24 h at 37°C. At the end of thisincubation, the cells were washed with 40 mMa-methyl-D-mannoside, suspended in fresh medium containing 10% FBS,and aliquoted into the wells of microtiter plates (1 x 105/well).After an additional 48-h incubation at 37°C with GSTat theindicated final concentration, [3H]thymidine incorporationwas assayed. During the final 48 h, cultures were supple-mented with either 5 ,ug/ml Con A or an equal volume ofmedium. Acpm indicates the difference in [3H]thymidine in-corporation between Con A-activated and control PBM. Mean[3H]thymidine incorporation of control PBMwas 1,121 cpm.This was not significantly affected by presence of GSTdur-ing the final 48 h of incubation.t Each term represents the mean±SEMof four separate ex-periments.

shown in Table IV, when varying concentrations ofGST were present in cultures of Con A-activated orcontrol lymphocytes during this latter 48-h incuba-tion, no significant inhibition of DNA synthesis wasobserved. If an additional 5-,ug/ml Con A was addedto the Con A-activated cells during the last 48 hof incubation, a marked increase in the degree of[3H Ithymidine incorporation was observed (Acpm= 95,995+2,589). This may have resulted from theinduction of a response in cells which had not beenactivated initially or perhaps by inducing daughtercells of the original Con A-activated population todivide again. In either event, GST caused concentra-tion-dependent inhibition of [3HIthymidine incorpora-tion induced by the addition of Con A to these cul-tures without affecting DNAsynthesis resulting fromthe initial Con A activation. These data indicate thatGST inhibition of lymphocyte proliferation does notresult from nonspecific toxicity or interference withthe assay system employed. Moreover, the observa-tion that the addition of GST after 24 h of mitogenactivation does not alter subsequent lymphocyteDNAsynthesis supports the concept that gold exertsits major influence during the initial induction phase oflymphocyte activation before DNAsynthesis actuallybegins.


Reversibility of GST inhibition of lymphocyte pro-liferation. PBMwere incubated with 5 ,ug/ml Con Aor an equal volume of medium as control for 24 hat 37°C in the presence or absence of variouis concen-trations of GST. At the end of this period, the cellswere washed with a-methyl-D-mannoside as pre-viously described and then washed four times withmedium to remove both excess Con A and GST.The cells then were suspended in fresh medium con-taining 10% FBS, aliquoted into the wells of micro-titer plates (1 x 105/well), incubated for an additional48 h at 37°C, and assayed for incorporation of [3H]-thymidine. As seen in Table V, the presence of GSTduring the period of activation resulted in no diminu-tion in subsequent [3H]thymidine incorporation if theinhibitor was removed after the first 24 h. These ob-servations suggest that the inhibitory effect of GSTdid not result merely from an alteration in the uptakeor handling of functionally relevant mitogen by PBM.

The inverse relationship between monocyte num-ber and GST inhibition of lymphocyte proliferation.Two sets of observations suggested the possibilitythat the effect of GST on mitogen and antigen re-sponsiveness might result from interference withmonocyte or macrophage function. First, the initiationof antigen- and mitogen-induced T-lymphocyte DNAsynthesis requires the active participation of an ac-

TABLE VGSTInhibition of Con A Responsiveness: Lack of Effect

when Present only during the Initial 24 H

GSTconcn.* during [3H ]Thymidine incorporation tCon A activation by Con A-activated PBM

pg/mnl Acpm

0 23,233+2,82425 20,828+ 1,797

100 21,881+7,829

* PBMwere incubated in test tubes (1 x 106/ml in mediumcontaining 10% FBS) with 5 ,ug/ml Con A or an equal volumeof medium as control and GSTat the indicated final concen-tration. After 24 h incubation at 37°C, the cells were exposedto 40 mMa-methyl-D-mannoside, washed four times, sus-pended in fresh medium containing 10% FBS, and aliquotedinto the wells of microtiter plates (1 x 105/well). After an addi-tional 48 h incubation at 37°C without GSTor additional mi-togen, [3H]thymidine incorporation was assayed. Acpm indi-cates the difference in [3H]thymidine incorporation betweenCon A-activated and control PBM. Mean [3H]thymidine in-corporation of control PBMwas 980 cpm. This was not sig-nificantly affected by presence of GSTduring the initial 24 hof incubation.t Each term represents the mean±SEMof three separate ex-periments. When GST was present during the entire 72 hincubation, 25 ,Lg/ml led to 25.8±5.4% inhibition and 100,ug/ml led to 65.1+3.9% inhibition of Con A responsiveness.

cessory cell (17-22). In populations of human PBMI,the monocyte stibserves this function (18, 21, 22).Second, GST has been demonstrated to be activelyinternalized by mnacrophages in vivo (29-31) and toinhibit certain enzyme systems (32), as well as ftunc-tional capabilities of these cells (33). Thus, the actionof gold on mitogen- and antigen-induced lymphocyteproliferationi might not result from inhibition oflymplhocyte responsiveness per se, but rather frominterferenice with the requisite function of the mono-cyte popuilation in the initiation or stupport of stuchresponses.

The magnittude of the proliferative response of Tlymphocytes induced by mitogens is dependent onthe number of accessory cells supporting that response(15, 20, 22). This accessory cell requirement is mosteasily observed in cultures of T lymphocytes whichhave been partially depleted of adherent cells. Instuelh cutlttures, the degree of mitogen-indtuced lympho-cyte [3H]thymidine incorporation varies directly withthe ntumber of accessory cells added. We reasoned,therefore, that if gold inhibited lymphocyte prolifera-tion by cauisinig a partial interference with a reqtuisitemonocyte ftunction, this inhibitory effect wouild beaccenittuated in ctulttures in which the nuimber of mono-cytes was decreased. Conversely, a GST-inducedmonocyte dysfuinction might be overcome by stupple-mentation of the cultures with excess monocytes.Fig. 4 depicts the relationship between the number ofml-onocytes stupporting a mitogen-induced proliferativeresponse and the degree of GST inhibition of thatresponse. GST inhibition of the Con A response ob-served in a standard population of PBM is shown.This popuilation of PBMcontained between 25 and30% monocytes. When the PBM were depleted ofadherent cells by sequential Petri dish plating and ny-lon coltumn passage, the resultant population contained0.5% monocytes. Two differences were observed inthe Con A response of the monocyte-depleted periph-eral blood lymphocytes (PBL) compared to PBM.First, the magnitude of the response in terms of Acpm(A) was diminished (from 72,606 to 18,396) and second,the degree of inhibition produced by all concentra-tions of GSTwas markedly greater (B). When the PBLwere supplemented with purified autologous mito-mvcin-C-treated monocytes to levels equivalent to thatfound in uinfractionated PBM (2.5 x 104 monocytes/1 x 105 PBL), the magnitude of the Con A responsewas restored to that observed with PBM. Likewise,the degree of GST inhibition was decreased and seento approximate that observed with PBM. When twiceas many monocytes were added to the culttures, GST-mediated inhibition of the Con A response wasftLrther decreased, although there was no furtherchange in the magnitude of [3H]thymidine incorpora-tion observed in cultures without added GST. The

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FIGURE 4 Con A-induced [3 H]thymidine incorporation as afutnction of GST concentration. PBM or PBL (1 x 105/well)wvere cuiltuired in flat-bottomed microtiter wells with or with-out 5 jig/ml Con A. PBL were cultured either alone orsupplemented with mitomycin-C-treated monocytes (2.5x IW~or 5 x 104I/well). Cultures also contained GST at finalconcentration indicated on the abscissa. After 72 h incubation,[3 H]thymidine incorporation was determined and expressedas Acpm (A) and percent inhibition of control stimulation (B).Each point represents the mean of triplicate determinations.When cuLltuired alone, the mitomycin-C-treated monocytepopulation manifested no significant degree of Con A-induiced [3 H]thymidine incorporation.

inverse relationship betweeni monocyte number andthe degree of GST inhibition of lymphocyte prolifera-tion stupports the contention that gold acts not byinhibiting the potential responsiveness of the lympho-cytes per se, but rather by interfering with a criticalftunction subserved by monocytes in these responses.

Effect of prolonged GST preincubation otn subse-qletnt lylynphocyte proliferation. Since previous ob-servations (Fig. 1) had suggested that decreasedlymphocyte proliferation might result from lengthyGST exposure, experiments were undertaken todetermine whether preincubating PBM with GSTwould alter their mitogen responsiveness. PBMwereincubated with GST for various lengths of time at37°C. The cells were then washed extensively, ali-quoted into the wells of microtiter plates, and incu-bated, with or without mitogen, for 72 h at 37°C infresh medium containing no gold. These latter cultureswere carried out at a variety of cell densities (1.25x 10J-1.O x 105/well) in microtiter plates with flat-bottomed wells. This modified limiting dilution cul-ture technique has been described previously (20)as a way to accentuate small differences in accessorycell number or function which might be missed instandard higher density cultures.

PBMwere cultured with varying concentrations of

Gold Inhibition of Lyrnphocyte DNASynthesis 461

E

z

8z

I

I

0

(22.8)

_ 0(47o(47.8)

(60.6)

o0' (59.7)

0(83.8)

/./ .

(856) s *-* Controls °--° GST-1i 0 g/ms GST-25 /Lg/ml

0o."" o GST-50O.Lg/ml0 (96.2)

1.25 2.5 5.0 10.0

NUMBEROF CELLSCULTUREDPER WELL(xlO-4)

FIGuRE 5 Effect of 72 h GSTpreincubation on subsequentCon A-induced lymphocyte proliferation. PBMwere incu-bated with GST at the indicated concentration for 72 h as

previously described. The cells were then washed, aliquotedinto flat-bottomed microtiter wells at various cell densities,incubated for 72 h with or without 5 ,ug/ml Con A, and assayedfor [3H]thymidine incorporation. Each point representsthe mean of triplicate determinations. Numbers in paren-

theses indicate percent inhibition compared to control.

GST for 72 h. Such preincubation of PBM in 10,ug/ml of GST had no significant effect on the abilityof these cells to respond to Con A when subsequentlycultured in the absence of the gold compound (Fig.5). However, a 72-h preincubation with higher con-

centrations of GST (25 or 50 jig/ml) rendered PBMdeficient in their ability to respond to Con A. Differ-ences in Con A responsiveness between these cellsand control cells became markedly more pronouncedwhen cultured at lower cell densities. These cells,likewise, were deficient in their responses to PHA,PWM, and SK/SD (data not shown). These findingscould not be explained by GST-induced cell deathsince the absolute number of cells surviving controland GST-containing cultures was not significantlydifferent and all cultures manifested similar viabilityas assessed by trypan blue exclusion after the 72 hincubation.

Development of GST-induced depression of mito-gen responsiveness required prolonged incubation.Thus, culture of PBM for 24 h with varying concen-

trations of GSThad no significant effect on the abilityof these cells to respond to mitogens when subse-quently cultured in the absence of gold (Table VI).Furthermore, limiting dilution analysis did not reveal


subtle differences resulting from GST preincubation(data not shown).

The data obtained from limiting dilution analysis(Fig. 5), suggested that gold-induced monocyte dys-function might explain the depressed responsivenessof PBMobserved after prolonged GST exposure. Forthis reason, the potential ability of fresh monocytesto restore mitogen responsiveness to PBMwhich hadbeen preincubated with GST was tested (Fig. 6).PBMwere preincubated for 5 days in varying concen-trations of GST. The cells were then extensivelywashed, aliquoted into the wells of microtiter plates,cultured for 72 h with or without Con A, and assayedfor [3H]thymidine incorporation. These latter cul-tures were supplemented with either fresh mediumas control, 2-ME, or fresh autologous monocytes.Those cells which had been preincubated with 10,ug/ml of GSTexhibited responses to Con A on subse-quent culture which were not dissimilar from controlcells which had been preincubated in medium alone.However, cells which had been preincubated withhigher concentrations of GST(25 or 50 ,ug/ml) showed amarked depression in Con A responsiveness whenrecultured alone. Addition of 2-ME to these culturesonly partially restored responsiveness. However, sup-plementation of these cultures with fresh autologousmonocytes resulted in a complete restoration oflymphocyte DNA synthesis to control levels. Thesedata suggest that prolonged exposure to GST had re-sulted in monocyte dysfunction and not an alteration inpotential lymphocyte responsiveness.

To confirm these observations, purified monocyteswere incubated for 72 h at 37°C with 10 or 25 ,ug/mlGST or an equal volume of medium as control.The cells were then washed extensively and evaluated

TABLE VIEffect of 24 H GSTPreincubation on Subsequent

Mitogen Responsiveness

Lymphocyte DNAsynthesisGSTconcni. duirinig24 h preincubation* () PHA Coni A PVVM

glhnl [3JH ]Thymidine incorporation-cpmn x 10-3

0 0.6±0.2 82.2±1.5 72.7±1.4 21.9±0.310 0.6±0.1 74.5±5.5 68.4± 1.1 22.5±0.425 0.6±0.1 85.1± 1.4 73.5±1.2 22.1±0.850 0.5±0.1 77.5±2.5 65.6+2.6 20.8±0.3

* PBMwere incubatedc in test tubes (1 x 106/ml) with GSTat various concentrations for 24 h at 370C. The cells werethen washed four times to remove GST, suspended in freshmedium, and aliquoted into microtiter wells (1 x 105/well).The cells were then incubated with 0.5 ,sg/ml PHA, 5 ,ug/mlCon A, or PWM(1:100) or an equal volume of medium ascontrol for 72 h, and assayed for [3H]Thymidine incorporation.I Each term represents the mean±SEMof triplicate deter-minations.

50 r

b 45

E 40'C0.

-35z0F- 30tr0a. 25

00z 20

wZ 15

10

EI

X' 5I')

0

NI 10 ID1 DI- PBMALONECM PBM+2-ME- PBMtMD(2.5xl04)EM PBM+MZ'(5x104)

01 111 10MEDIUM GST-Kag/mI GST-25pg/ml GST50Ig/ml

5 DAY PREINCUBATION

FIGURE 6 Monocyte rescue of GST-induced depression ofCon A responsiveness. PBMwere preincubated with variousconcentrations of GST for 5 days at 37°C. The cells werethen washed four times, suspended in fresh medium contain-ing 10% FBS, aliquoted into flat-bottomed microtiter wells(2.5 x 104/well), incubated with or without 5 ug/ml Con Afor 72 h at 370C, and assayed for [3H]thymidine incorporation.These latter cultures were supplemented with eithermedium as control, 25 ,uM 2-ME or fresh autologous mono-cytes (Mb). The monocytes had been treated with mito-mycin-C and manifested no significant [3H]thymidine in-corporation in response to Con A when cultured alone. Eachbar represents the mean of triplicate determinations.

for their ability to support mitogen responsiveness inpopulations of fresh PBL which had been depletedof adherent cells (Fig. 7). When fresh adherent cell-depleted PBL were cultured alone, no significantdegree of DNA synthesis was triggered by Con A.Supplementation of these cultures with aged controlmonocyte or macrophage (Mb) led to the developmentof a vigorouis proliferative response to Con A. By con-trast, Mb, which had been preincubated for 72 h withGST, were markedly deficient in their ability to supportCon A-induced T-lymphocyte proliferation. This de-ficiency could not be explained by cell death, since cellyields, both in terms of absolute number and viabilityas gauged by trypan blue exclusion, were similar inall three monocyte populations. Furthermore, it isunlikely that these observations resulted from carryover of GST or induction of a suppressor cell sinceGST-preincubated monocytes did not inhibit Con A-induced DNAsynthesis of PBL supported by controlmonocytes (data not shown).

DISCUSSION

A number of clinical trials have established thatchrysotherapy can lead not only to amelioration of thesymptoms of rheumatoid arthritis but also to actualremissions of disease activity (10-13). Since the patho-

genesis of chronic rheumatoid synovitis is thought toinvolve immunologic mechanisms (1), it seems rea-sonable to suggest that the therapeutic efficacy of goldcompounds may relate to their ability to modify im-mune responsiveness. Although a number of studieshave indicated that gold compounds may inhibit non-specific inflammatory responses in a variety of experi-mental models (33-35), evidence as to whether theseagents exert a direct effect on lymphocyte responsive-ness remains conflicting (36).

To conclude that the effects of gold compounds ob-served in vitro have significance in vivo, such effectsshould be observed with concentrations attainablein treated patients. A number of studies have quanti-tated the levels of gold present in various bodyfluids and tissues after therapy with different goldcompounds. Although blood concentration of goldhas been shown to depend on variables such as dosageschedule and excretion rate, serum levels attained inpatients treated wvith gold tend to be in the range of2-5 ,ug/ml (37-42), equivalent to 4-10 gg/ml of GST.

50'"-*so~~~~~~~~~~~- ....I -..0

;; Control --o~~~ - o---l-

x 40 l0

06i230 / ~~~~GST-IClWg /ml

II/

z Io

~30a-0 /z

20

5 06I.5 5 1

0.I

SUPPLEMENTALMONOCYTES(x10-4)

FIGuRE 7 The effect of GSTpreincubation on the ability ofmonocytes to support Con A-induced proliferative responses.Purified monocytes were suspended in medium containing10% FBS (0.5 x 106/ml), and cultured in test tubes with orwithout GST. After a 72-h incubation at 37°C, the cells werewashed three times, exposed to 40 ,tg/ml mitomycin-C for60 min at 37°C, and washed four more times. They werethen mixed with fresh, autologous, adherent-cell depletedPBL (2.5 x 104/well) in flat-bottomed microtiter wells andincubated with 5 ,ug/ml Con A or an equal volume of mediumas control. After a 72-h incubation at 37°C, [3H]thymidineincorporation was determined. Each point represents themean of triplicate determinations. Con A-induced [3H]-thymidine incorporation manifested by PBL cultured aloneresulted in Acpm = 248. The monocytes, when culturedalone, made no significant response to Con A.


However, the concentration of gold in the blood doesnot appear to correlate with either the therapeuticor toxic effects of gold compounds in man (38-42),suggesting that the anti-inflammatory action of goldcompounds is not related to serum gold level. Theadministration of gold compounds leads to rapiddistribution of gold throughout the body with atendency for it to concentrate in areas of inflamma-tion as well as in organs rich in reticuloendothelialelements (43,44). Thus, tissue gold levels may pro-vide a more meaningful estimation of both pharma-cologically attainable and therapeutically effectivegold concentrations. Mean synovial tissue gold con-centrations of 21.1 ,ug/g of tissue (wet weight) havebeen reported in a group of 14 patients recentlytreated with a mean total dose of 1.84 g of gold (45).These findings are consistent with those reported in apatient who had received a total gold dose of 2,530mg over a 5-yr period and at postmortem examinationhad a mean synovial tissue gold concentration of 25gg/g wet weight (46). Although such estimations oftissue concentrations obtained by atomic absorptionspectroscopy give no information about the physicalstate of the gold or its availability to cellular elementsin the synovium, these data suggest that the maximalconcentration of gold attainable at the relevant tissuesite is equivalent to 42-50 0g/ml of GST.

GST was found to reversibly inhibit antigen- andmitogen-induced human lymphocyte proliferation.These findings are similar to results reported byothers using PHA(47, 48) and allogeneic lymphocytes(48) as stimuli. Inhibition of responsiveness wasdependent on the concentration of GST in the culturewith significant suppression observed well within therange of serum and tissue levels found in patientstreated with gold compounds. Inhibition of lympho-cyte proliferation was dependent on the gold ionitself and not the sulfur-containing ligand inasmuch asGST and gold chloride were both inhibitory whileTMAwas not. Suppression of responsiveness couldnot be ascribed to cell death or an alteration in thekinetics of the in vitro response. Moreover, goldinhibition of responsiveness did not result from aninterference with the binding or handling of mitogenby the cells. This was indicated by the observationthat GSTpresent only during an initial 24-h period ofmitogen activation, but not during the subsequent 48 hof incubation, had no effect on the magnitude of theresultant proliferative response. This was supported bydata obtained when the binding of radioiodinatedmitogens to human PBM was studied. 100 ug/mlGST had no effect on the binding of either 125I-PHAor 125I-Con A measured after a 30-min incubation at40C (unpublished observation).

Gold compounds were found to act by specificallyinterfering with an early step in the inductive phaseof lymphocyte activation, even before DNAsynthesis

began. Initiation of T-lymphocyte proliferation by thestimulating agents used requires the active participa-tion of an accessory cell, which itself does not undergoa DNAsynthetic response (17-22). In populations ofhuman PBM, the monocyte subserves the requisiteaccessory cell function (15, 18, 19, 21, 22). Therefore,experiments were carried out to determine whetherone or the other participant in this two-cell respond-ing unit was differentially affected by gold compounds.This question was first approached by investigatingthe inhibitory capacity of GST in cultures with variousratios of responding to accessory cells. An inverserelationship was noted between the degree of GSTinhibition of the proliferative response to mitogensand the number of monocytes supporting that response.The data suggested that the action of GSTon lympho-cyte activation resulted not from inhibition of thepotential responsiveness of the T-lymphocyte popula-tion, but rather from interference with the ability ofthe monocytes to serve as effective accessory cells inthe initiation of such responses. Alternatively, mono-cytes merely could have acted in a protective capacityin these experiments, perhaps by internalizing GSTand thus preventing its inhibitory effect on lympho-cytes. To investigate this possibility, 100 jig/ml GSTwas incubated with PBM(containing 30% monocytes)or with medium alone for 24 h at 37°C, and then testedat various concentrations for its capacity to inhibitlymphocyte proliferation. Preincubation of GST withPBM (1 x 106/ml) did not diminish its subsequentinhibitory action on Con-A-induced lymphocyteDNAsynthesis compared to GST similarly incubatedin medium alone (unpublished observation). Thesedata militate against the possibility that the functionof monocytes in the cultures was to protect lympho-cytes from the effects of gold by internalizing orotherwise inactivating GST.

Another indication of the cellular site of action ofGST became apparent only after prolonged incuba-tion of PBM with this gold compound. Thus, pre-incubation of PBM with GST for 72 h resulted indiminished mitogen responsiveness of these cells onsubsequent challenge. This effect could be inducedby prolonged exposure to concentrations of GST (25or 50 ,ug/ml) equivalent to those found in the syno-vial tissues of patients treated with gold compounds.Furthermore, this effect was irreversible inasmuch asthe PBM exhibited diminished mitogen responsive-ness even after removal of GST. The ability of freshautologous monocytes to restore mitogen responsive-ness to these populations indicated that prolongedexposure of this mixed PBMpopulation to GST hadnot affected the intrinsic ability of the lymphocytesto respond but rather had altered the functional capac-ity of the monocytes to support such responses.This interpretation was confirmed by the observationthat preincubation of purified monocytes with GST


for 72 h rendered them deficient in their ability tosupport mitogen-induced T-lymphocyte prolifera-tion on subsequent culture.

These studies indicate that a major action of goldcompounds involves interference with the functionalcapability of Mb. This conclusion is consonant with anumber of previous observations. First, microscopeexamination of rheumatoid synovial tissue obtainedfrom patients treated with gold compounds has re-vealed a selective accumulation of gold particles inthe lysosomes of the type A synovial cells and othermacrophages of the synovium (29-31). Second, GSThas been shown to be actively endocytosed in vitroby guinea pig peritoneal macrophages with resultinginhibition of their lysosomal enzyme activity (32).Finally, the phagocytic activity of macrophages inexperimentally induced inflammatory exudates hasbeen found to be suppressed in patients with rheu-matoid arthritis receiving gold therapy as comparedto untreated rheumatoid patients (33).

Mononuclear phagocytes play a central role in theinitiation and maintenance of chronic inflammatoryreactions. They not only function as effector cells insuch responses (49), but also possess the uniquecapacity to interact with both B and T lymphocytesin the induction of cellular and humoral immuneresponses. Thus, antigen-induced proliferation andmediator synthesis by T lymphocytes require the ac-tive participation of M1D (17, 50). These accessorycells initially take up antigen and act to facilitate itsfunctionally effective presentation to primed Tlymphocytes (51). Similarly, macrophages play a criti-cal role in the humoral response to many antigens bysupporting the induction of antigen-specific helperT cells (52) and providing a focus for collaborationbetween helper T cells and B cells whose progenysecrete antibody (53, 54). In view of the critical role ofmononuclear phagocytes in the induction of bothcellular and humoral immune responses, pharma-cologically induced interference with their functionby gold compounds may, in part, explain the efficacyof these agents in reducing the chronic immunologi-cally mediated inflammation of rheumatoid arthritis.

ACKNOWLEDGMENTSWewish to thank Ms. Zora Dugan for her expert technicalassistance and Ms. Terrie Harris and Ms. Bonita Walkerfor preparing the manuscript.

This work was supported by U. S. Public Health ServiceProgress grant AM-09989.

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466 P. E. Lipsky antd M. Ziff

inhibition of antigen- mitogen-induced lymphocyte proliferation … · adherent cells; pbm,...

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