Differential inhibition of human T-lymphocyte activation by maleimide probes

CELLULARIMMUNOLOGY 101,181-194

(1986)

Differential Inhibition of Human T-Lymphocyte by Maleimide Probes’ BRIAN

Activation

M. FREED,BOBAKMOZAYENI, DAVID A. LAWRENCE, FRANCES R. WALLACH, AND NEIL LEMPERT

Department of Surgery and the Department of Microbiology and Immunology, Albany Medical College of Union University, Albany, New York 12208 Received December 9,198s; accepted March 25, 1986

Cellular thiols are known to be involved in lymphocyte activation, differentiation, and growth. In theory, alkylation of selective cellular thiols could be used to regulate specific processesin the activation sequenceby inactivating particular enzymes or structural proteins, although to date specific alkylating probes have not been reported. N-Ethylmaleimide (NEM) is a lipophilic sulfhydryl-alkylating agent that is known to block the in vitro proliferative response of T lymphocytes. NEM (10 PM) was found to be fully inhibitory in PHA, Con A, and MLC assaysonly when added prior to or simultaneously with the mitogens or allogeneic cells; the addition of NEM only 15set after stimulating the cells with PHA resulted in a loss of >50% of the inhibitory activity. The addition of 50 &42-ME 10 min after treating the ceils with NEM failed to block the inhibitory effect. NEM (LO-20 PM) had no adverse effect on lymphocyte viability, but completely blocked lymphocyte agglutination in responseto mitogens or allogeneic cells. The lymphocytes overcame the inhibitory effectsof NEM after 48 hr in both the PHA and MLC experiments. Resumption of the proliferative responsewas associatedwith the onset of agglutination in the PHA assay. In experiments using various analogs of NEM, we noted that the presence of a nonpolar N-linked side group was necessary for inhibitory activity. Pretreatment of PBMC with NEM decreasedthe total cellular thiols by 50% and blocked proliferation by 99%, whereas N-hydroxymaleimide decreasedthe total cellular thiols by 38%but had no effect on the proliferative response. The additional 12% of the cellular thiols that react with NEM, but not NHM, account for the inhibitory effect of NEM on lymphocyte proliferation. These findings suggest that selective cellular thiols are critical for T-cell activation. 0 1986 Academic press. Inc.

INTRODUCTION Cellular thiols are well known to be involved in many cellular processes,such as transport acrossthe cell membrane (l-3) DNA synthesis (4,5), protein secretion (6, 7) receptor-ligand interaction (8, 9), movement (lo- 12), and numerous enzymatic activities ( 1, 13, 14). Lymphocytes may be more sensitive to thiol modulation than many other cell types, as evidenced by their interphase sensitivity to ionizing radiation ( 15). Lymphocyte activities are modulated by drugs ( 16, 17) and endogeneous factors (18, 19) that react with thiols. In addition, treatment of lymphocytes with ’ This work was supported by the James Hilton Manning and Emma Austin Manning Foundation and by NIH Grants ES 03778 and BRSG S07RR05394-23. B.M. and F.R.W. are Physician-Investigator Research Fellows whose work was supported by a grant from the Pew Memorial Trust. 181 0008-8749186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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thiol-blocking agents has been shown to inhibit membrane transport phenomena (3, 13), interfere with mitosis by depolymerizing tubulin (20), and modify various lymphocyte effector functions (2 1). Although modulation of lymphocyte cellular thi01shas been shown to alter particular functions, few attempts have been made to correlate particular cellular thiols with specific biochemical events in lymphocyte activation, differentiation, and growth. In order to do this, thiol-reactive probes with different specificities need to be identified. Maleimide probes have been used for many years to study the involvement of thiols in the functional activity of a variety of proteins (22, 23). They are relatively stable reagents and have been shown to react stoichiometrically with thiols (24). N-ethylmaleimide (NEM)’ is a lipophilic maleimide that has been shown to block the in vitro proliferation of T lymphocytes in response to phytohemagglutinin (PHA) (20, 25, 26). The inhibitory effect of NEM is manifest initially as a block in the agglutination of PHA-stimulated cells (20,27), although NEM does not interfere with the binding of the mitogen to the cells (25). It has been suggestedthat NEM inhibits mitogenesis by blocking the polymerization of microtubules (MT) (20). However, NEM is known to block the activity of guanylate cyclase (28), adenylate cyclase (29), DNA polymerase (30), and other enzymes (1) by reacting with important sulfhydryl groups, so it is possible that the inhibition produced by NEM is due to its effects on more than one event in the process of lymphocyte activation and proliferation. The fact that maleimides exhibit a million-fold greater reactivity with thiols compared to amino groups at physiological pH (31) makes these compounds useful as probes for studying the importance of thiol-dependent events in the T-cell activation process.The purpose of these studies was to utilize maleimide probes of different size and polarity to determine the effects of differential thiol blockage on the activation of human T lymphocytes. MATERIALS AND METHODS Reagents. Diamide (diazine dicarboxylic acid bis[N,N-dimethylamide]), Con A (concanavalin A), showdomycin (3-/3-D-ribofuranosylmaleimide), and 2-ME (2-mercaptoethanol) were obtained from Sigma Chemical Company (St. Louis, MO.). NEM (N-ethylmaleimide), NMM (N-methylmaleimide), maleimide, NPM (N-phenylmaleimide), NHM (N-hydroxymaleimide), A25,335-9 (Cmaleimido-TEMPO), A25,337-5 (3-maleimido-PROXYL), A25,338-3 (3-(maleimidomethyl)-PROXYL), A25,339-1 (3-(2-maleimidoethylcarbamyl)-PROXYL), A25,340-5 (3-(3-maleimidopropylcarbamyl)-PROXYL), and A25,341-3 (3-(2-[2-maleimidoethoxyll-ethylcarbamyl)-PROXYL) were obtained from Aldrich Chemical Company (Milwaukee, Wise.). PHA-M (phytohemagglutinin HA- 15) was purchased from Wellcome Diagnostics (Dartford, England). Cells. Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized whole blood from normal donors by density gradient centrifugation on Fi* Abbreviations used: PBMC, peripheral blood mononuclear cells; HBSS, Hanks’ balanced salt solution; BSS,balanced salt solution; PBS, Delbecco’s phosphate-buffered solution; PHA, phytohemagglutinin; Con A, concanavalin A; [‘H]TdR, tritiated thymidine; MLC, mixed leukocyte culture; MLR, mixed leukocyte response;CTL, cytotoxic T lymphocytes; AGHS, agamma human serum; SCR, specific chromium release; MT, microtubule; 2-ME, 2-mercaptoethanol; TEMPO, 2,2,6,6-tetramethyl-I-piperidinyloxy, free radical; PROXYL, 2,2,5,5-tetramethyl- I-pyrrolidinyloxy free radical.

MALEIMIDES

DELAY

T-CELL

RESPONSE

183

toll-Paque (Pharmacia, Uppsala, Sweden). The PBMC were removed from the Fitoll-plasma interface and washed three times in Hanks’ balanced salt solution (HBSS), and then resuspended to lo6 cells/ml in RPM1 1640 (MA Bioproducts, Walkersville, Md.) supplemented with 100 units/ml penicillin, 100 pg/ml streptomycin, and lo-20% heat-inactivated human AB serum (Flow Laboratories, Mclean, Va.). Mitogen assays. PBMC (2 X lo5 cells in 0.2 ml vol) were cultured in 96-well flatbottom tissue culture plates (Costar) in RPM1 1640 supplemented with 10% AB serum. The cells were stimulated with 250 pg/ml PHA-M or 10 pg/ml Con A and incubated at 37°C in 5% COJ95% air for 3 days unless otherwise indicated. Eighteen hours before terminating the culture, each well was pulsed with 0.6 PCi of tritiated thymidine ( [3H]TdR; Schwarz-Mann, Spring Valley, N.Y., sp act = 1.9 Ci/mmol). The cultures were then harvested onto glassfiber filters with a Mash II cell harvester (MA Bioproducts). The filter disks were placed in scintillation vials with Econofluor (NEN Research Products, Boston, Mass.) and counted in a liquid scintillation counter (Beckman Instruments, Irvine, Calif.). The data are expressed as means +- standard deviation of counts per minute (cpm)/culture of t3H]TdR uptake. Mixed leukocyte culture (MLC). Unidirectional MLCs were performed by co-culturing 1O5responder PBMC with lo5 irradiated (3000 rad, cesium source) allogeneic stimulator PBMC in RPM1 1640 supplemented with 20% human AB serum. The cells were plated in 96-well flat-bottom plates and incubated for 6 days at 37°C in 5% CO,/95% air. The wells were pulsed and harvested as described for the mitogen assays.The data are expressedas experimental (allogeneic) minus control (autologous) in counts per minutes of [3H]TdR uptake. Agglutination response.Agglutination of PBMC in PHA assayswas determined by daily viewing of the cultures under 200X phase-contrast magnification using a Nikon TMS microscope. The degree of agglutination was scored from 0 to 5 as follows: 0 = no agglutination, 1 = 20% agglutination, 2 = 40% agglutination, 3 = 60% agglutination, 4 = 80% agglutination, 5 = 100%agglutination. PHA responsesalways exhibited 80- 100%agglutination by Days 2-3 while unstimulated cells exhibited no agglutination during the first 7 days in culture. Generation of cytotoxic T lymphocytes (CTL). PBMC from two normal donors were resuspended to lo6 cells/ml in RPM1 1640 supplemented with 20% AB serum. Unidirectional MLCs were then performed by co-culturing lo6 responder cells with lo6 irradiated stimulator cells in a final volume of 2 ml in 24-well tissue culture plates (Costar). Syngeneic MLCs were performed at the same time and served as controls. The plates were incubated for 10 days as described for the MLC. Generation of PHA blasts. PHA blasts to be used as target cells were generated by culturing 2 X 1O6cells in RPM1 1640 supplemented with 10%agamma human serum (AGHS; KC Biologicals, Lenexa, Kans.) and 250 pg/ml PHA-M in a final volume of 2 ml in 24-well tissue culture plates (Costar). Three days later, the blasts were isolated, washed three times in HBSS, and resuspended to 5 X lo5 cells/ml in RPM1 1640 supplemented with 10% AGHS and 10% interleukin-2 (Cellular Products, Buffalo, N.Y.). The cells were cultured for 3 days, after which the medium was replaced and the cells cultured for an additional 2 days. These cells were then washed three times in HBSS and resuspended to 5 X lo6 cells/ml in RPM1 1640 (no serum). The cells were incubated with 250 &i of 5’Cr (NEN Products, Boston, Mass.) for 1 hr at 37°C and then washed three times in HBSS and 5% AGHS at 4°C. The cells were then

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ET AL.

resuspended to 1O5cells/ml in RPM1 1640 + 10%AGHS and chilled on ice until they were used in the CTL assay. CTL assay. CTL effector cells were harvested on Day 10 and washed three times in HBSS. The cells were resuspended to 5 X lo6 cells/ml in RPM1 1640 and 10% AGHS. PHA blasts ( 100 ~1( 1O4cells)) and 100 rl(5 X 1O5cells) of effector cells were placed in 96-well V-bottom plates. The plates were centrifuged at 200g for 10 min, and then incubated for 4 hr at 37°C in 5% C02/95% air. After the 4-hr period, the control wells were treated with 100 ~1of RPM1 1640 (0% lysis) or I N NaOH (100% lysis). The plates were centrifuged at 300g for 10 min at 4°C. Supernatant ( 100 ~1) was removed from each well, placed in 12 X 75-mm glass tubes, and counted in a gamma counter (Beckman). Each assayconsisted of three to six data points. Specific chromium release(SCR) was calculated by the equation SCR = {[(A - B) - (A - A)]#&,

- C,]} X 100,

where (A - B) is the cpm of “Cr released by A + Bx effector cells incubated with B target cells, (A - A) is the cpm released by the same effector cells incubated with autologous (A) target cells, Coo is the 100% lysis control, and C,, is the 0% lysis control. Assayfir total cellular thiols. Total cellular reduced sullhydryls were quantitated by the procedure of Ayers et al. (32). PBMC were resuspended in PBS and plated into 96-well microplates at 1O5cells/well. Each well was treated with 2 mg/ml of the thiolspecific fluorochrome, N-(4-[7-diethylamino-4-methylcoumarin]-3-yl)phenylmaleimide (Molecular Probes, Junction City, Oreg.), which fluoresces upon alkylation of a reduced sulfhydryl. The plate was incubated in the dark at room temperature for 1 hr and then read on a Dynatech Microfluor Reader. The data are expressedas relative fluorescence units. Flow cytometric analysis of lymphocyte subpopulations. A Coulter Epics V was used for the analysis of cultured cells. The instrument was operated with an argon ion laser at 488 nm and 300 mW. The cells were gated by forward angle light scatter and 90“ light scatter. Viable cells were gated by propidium iodide exclusion. The lymphoid population was analyzed for green fluorescence emitted by fluorescein conjugated to monoclonal antibodies specific for T3 (CD3), T4 (CD4), and T8 (CD8) (Ortho Pharmaceuticals, Raritan, N.J.). The background fluorescence was quantitated for nonspecific binding of mouse IgG with the use of fluorescein conjugated to mouse IgG that lacked nonspecific binding to human cells. Immunofluorescent staining and analysis were performed asdescribed (33,34). Results represent the analysis of 10,000 cells. RESULTS Kinetics ofNEM-mediated inhibition ofMLC and PHA responses.NEM (10 PM) blocked the MLC and PHA responsesby >95% when added simultaneously with the allogeneic cells or mitogen (Fig. 1). The addition of NEM 15 min after initiating the MLC resulted in 46% inhibition of [3H]TdR uptake, and NEM added after 6 hr was not inhibitory. The effect of NEM on the PHA response was even more striking. The addition of NEM even seconds after PHA resulted in ~50% inhibition, and the addition of NEM after 1 hr was not inhibitory (Fig. 1B). In contrast, the membranepermeable, thiol-oxidant diamide at 0.2 ticompletely blocked the MLC and PHA

MALEIMIDES

DELAY T-CELL RESPONSE

185

FIG. 1. NEM inhibits very early events in the T-cell proliferative response. NEM (0) and diamide (0) were added to MLC (A) and PHA (B) assayssimultaneously with or at various times alter the addition of either allogeneic cells or the mitogen. The final concentrations of NEM (10 pLM) and diamide (200 PM) had been shown to be the minimal concentrations that produced >95% inhibition of the MLC and PHA responses.The shaded areas represent the means f SD of the control responses.

responsesregardlessof when it was added. Both of these agentswere used at the lowest concentrations that were found to produce >95% inhibition in PHA, Con A, and MLC assayswithout decreasing cell viability. In order to determine how long NEM had to be in contact with the cells in order to block the mitogenic response, 2-ME was added to the culture simultaneously with NEM or 10 min after PHA stimulation. 2-ME reacts stoichiometrically with NEM in less than 10 set (data not shown), so the addition of fivefold excess 2-ME was considered sufficient to inactivate unreacted NEM molecules. As can be seenin Table 1, the addition of 2-ME at T = 0 completely abolished the inhibitory effect of NEM. However, the addition of 2-ME 10 min after PHA and NEM failed to block the inhibition. These data suggestthat NEM reacts quickly with the cells and its continued presencebeyond the first few minutes is not necessary.To test this hypothesis further, PBMC were pretreated with 10 PM NEM for 30 min at 37°C washed, and assayed for PHA and Con A responsiveness(Table 2). Although this pretreatment resulted in 82% inhibition of the PHA responseand 93% inhibition of the Con A response,com-

TABLE 1 NEM Inhibition Occurs within Minutes NEM

2-ME”

[3H]TdR uptake (cpm)

+ + +

+ + (T=O) +(T= IOmin)

101,386 + 8425 104,221 f 5888 1,434 f 1095 105,607 + 62 1I 1,217+ 222

’ 2-ME (50 &4) was added to the wells with NEM (10 pM), but the two were not mixed prior to the addition of the cells.

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FREED ET AL. TABLE 2 Effect of NEM Pretreatment on Mitogenesis [‘H]TdR uptake (cpm)

Cells

PHA

% Inhibition

Con A

% Inhibition

Control Pretreated NEM(T= 0)

132,789 f 3678

-

56,733 k 638

-

23,552 f 2157

106+

51

82 99

3,706 x!z762 74k 18

93 99

Note. PBMC were treated at lo6 cells/ml for 30 min with 10 p,V NEM, washed three times in HBSS, and resuspended in RPM1 1640 + 10% AB serum. NEM was added directly to the wells at T = 0 in other cultures. Both treatments resulted in >99% viability as determined by trypan blue dye exclusion.

plete inhibition of the mitogenic responses(99% inhibition) was observed only when NEM was added to the culture at T = 0. Reversibility of NEM inhibition. The fact that 2-ME failed to reverse the NEMmediated inhibition was not surprising in light of the known stability of the thiol ether adduct. However, when the kinetics of proliferation in control and NEMtreated cultures were examined, we noted that the inhibition was spontaneously and completely reversed (Fig. 2). In both MLC and PHA assays,10 PMNEM consistently delayed the peak proliferative responsesby 48-72 hr. It is interesting to note that 20 &4 NEM had no inhibitory effect on proliferation when added 2 hr after PHA, whereas 20 PM NEM added prior to PHA resulted in permanent inhibition of the proliferative response (data not shown). Furthermore, the addition of 50 PM 2-ME or exogeneous interleukin-2 (IL-2) 1 hr after NEM did not alter the shift in kinetics. To determine if the spontaneous reversal of NEM inhibition was a function of activated cells alone, or if quiescent cells were also capable of recovering from the inhibitory effects, PBMC were cultured in the absence or presence of 10 j.J4 NEM and stimulated with PHA O-5 days later. The inhibitory effect of NEM gradually decreasedover the 5-day period (Table 3). However, it should be noted that the control response was 32% lower when the mitogen was denied for 5 days. In agreement with Pfeifer and Irons (20, 27), we observed that NEM completely blocked the agglutination of PBMC in response to PHA. It was therefore interesting

DAYS FIG. 2. NEM shifts the kinetics of T-cell proliferation. NEM (10 PM) was added to MLC (A) and PHA

(B) assaysat T = 0. Wells were pulsed daily with [3H]TdR and harvested 18 hr later (same day). Peak proliferative responseswere shifted to the right by 2 days in the presence of NEM (0) compared to the control (0). Data are expressedas means -CSD.

MALEIMIDES

187

DELAY T-CELL RESPONSE TABLE 3

Reversal of NEM Inhibition in Quiescent PBMC Day PHA added

Treatment”

Agglutination b

i3H]TdR uptake (cpm)

0

Control NEM Control NEM Control NEM Control NEM Control NEM Control NEM

5 1 4 3 4 3 4 4 4 4 3 3

46,333 f 1679 1,478 -+ 1602 41,892 k 4725 10,095 + 4303 42,455 f 4666 12,084 + 384 40,560 f 1846 18,41I f 10278 36,792 + 1291 27,133 zk 10752 31,594? 2513 24,801 f 1385

1 2 3 4 5

(1PBMC were placed in flat-bottom tissue culture plates in media plus 10% AB serum. NEM (10 p1I4) was added prior to the mitogen on Day 0. PHA was added on the days shown and the wells were pulsed with i3H]TdR 48 hr later. bAgglutination was recorded on the day the wells were harvested.

to note that the spontaneous reversal of NEM-mediated inhibition was associated with the onset of agglutination, which was also delayed by 48 hr (Table 4). Generation of CTL in the presence of NEM. Our data suggestedthat NEM delays the activation process, but it was considered possible that the late [3H]TdR uptake observed in the PHA and MLC assayswas due to proliferation of a separate subset

TABLE 4 Effect of NEM on Leukocyte Agglutination and PHA Responsiveness

Day

Treatment’

Agglutination

[3H]TdR uptake (cpm)

1

Control NEM Control NEM Control NEM Control NEM Control NEM

1 0 4 1 5 2 5 4 b 5

15,096 f 2308 146? 158 38,410 + 1606 1,349* 141 120,417 + 6561 14,774 f 1142 111,400~8910 76,428 f 2729 13,745 f 1220 118,625 f 247

2 3 4 7

a Cells were treated with PHA on Day 0. Agglutination was scored at the beginning of the 18-hr pulse period. Data are expressedas means + SD. ’ Agglutination was measured as described under Materials and Methods. After 4 days in culture, the PHA blasts no longer remain in the clusters.

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FREED ET AL.

FIG. 3. Effect of NEM on the MLR and generation of CTL. The MLR was measured on Day 5 by pulsing the plate for 18 hr with [3H]TdR. The generation of CTL was assayed on Day 10 as described under Materials and Methods. The figure shows the responsesin the absence (solid bars) and presence (slashed bars) of 10 pM NEM. The left-hand ordinate represents the MLR and the right-hand ordinate represents CTL activity.

of T cells. We therefore performed an experiment in which the MLC was used to generate CTLs. The addition of 10 PM NEM blocked the proliferative response(measured on Day 5) by 92.5%, but the cytolytic activity (measured on Day 10) in NEMtreated (22.2% SCR) and control cultures (27.6% SCR) was not significantly different (Fig. 3). Furthermore, the phenotypes of control (CD3 = 81%, CD4 = 49%, CD8 = 30%) and NEM-treated cells (CD3 = 84%, CD4 = 56%, CD8 = 28%) were not substantially different. These data suggestthat NEM delays the T-cell activation process in the MLC without affecting the generation of cytotoxic T cells and without apparent alteration of the lymphoid subsets. Inhibition ofPHA responsivenessby NEManalogs. The maleimide alkene is highly susceptible to nucleophilic attack by sulfhydryl groups and thus confers thiol reactivity on the molecule. The N-linked maleimide side chains have no effect on thiol reactivity per se. However, the side chains may alter lipophilicity and can cause steric hindrance, thereby altering the biological effectsof the maleimides. In order to determine if maleimide N-substitutions could alter maleimide inhibitory activity, various analogs of NEM were tested in the PHA assay.As can be seen in Fig. 4, substituting the ethyl group with a methyl group (NMM) diminished the inhibitory effect by nearly 50%; substituting the ethyl group with a hydrogen (maleimide) diminished the inhibition by approximately 85%. Substituting the ethyl group with phenyl (NPM), TEMPO (A25,335-9) or PROXYL (A25,337-5) rings did not diminish the suppressive activity, and the presence of an additional methyl group between the maleimide and PROXYL moieties (A25,338-3) had no appreciable effect on inhibitory activity. The presence of an ethylcarbamyl (A25,339-1) or a propylcarbamyl (A25,340-5) group between the maleimide and PROXYL moieties reduced the inhibitory activity to ~10% of the parent compounds. No inhibitory activity could be detected when the maleimide and PROXYL moieties were separated by an ethoxyethylcarbamyl group (A25,34 l-3). The polar N-hydroxymaleimide (NHM) was found to be inactive, as was the maleimide-containing (not N-linked) antibiotic, showdomycin. NPM, A25,335-9, A25,337-5, and A25,338-3 all exhibited the characteristics of NEM-mediated inhibition. Specifically, these maleimides were all inhibitory at 8- 10 PM, had to be present at T = 0 in order to block proliferation, blocked PHA-induced agglutination, and only temporarily blocked the activation process. Ejkt of NEM and NHM on total cellular thiols. NEM is a lipophilic molecule (35) while NHM is hydrophilic. It is therefore possible that NHM lacks inhibitory activity

MALEIMIDES

Analog

189

DELAY T-CELL RESPONSE

Structure

Percent

Inhibition

N.ethylmaleimide

99

N. methylmaleimide

52

Maleimide

15

N .phenylmaleimide

99

N - hydroxymaleimide

0

A25,335-9

98

A25.337.5

A25,338 - 3

A25,339-1

98

3

A25,340- 5

10

A25,341-3

0

Showdomycin

0

FIG. 4. Effect of NEM analogs on PHA responsiveness.The maleimides were tested at 5-100 &f. NMM exhibited 99% inhibition at 20 &and maleimide exhibited 99% inhibition at 50 &f. NHM, A25,339-1. A25,340-5, A25,341-3, and showdomycin exhibited < 10%inhibition at concentrations up to 100 PM. For the sake ofcomparison, only the percentage inhibition at 10PMis shown.

because it is taken up slowly by the cells (allowing more time to react with serum thiols) or because it cannot react with critical cellular thiols once inside the cell. To test these hypotheses, PBMC were treated with 10 PM NEM or NHM in either BSS or RPM1 1640 + 10% AB serum. The cells were then washed and assayedfor total cellular thiols and the ability to respond to PHA. As can be seen in Fig. 5, NEM in BSS decreased the total cellular thiols by 50% while NHM decreased the thiols by 38%. Under these conditions, NEM blocked the PHA response by >99%, but NHM

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FREED ET AL.

NEM + BSS

NHM + BSS

NEM + MEDIA

NHM t MEDIA

FIG. 5. Effect of NEM and NHM on total cellular thiols. Peripheral blood mononuclear cells were treated with 10 pM NEM or 10 PM NHM in BSS or RPM1 1640 + 10% AB serum (medium). The cells were treated for 30 min at 37°C and then washed twice in BSS. A portion of the cells was then resuspended in RPM1 1640 + 10%AB serum and assyed for PHA responsiveness(data are expressed as cpm of [3H]TdR uptake in thousands). The remainder of the cells were resuspended in PBS at lo6 cells/ml and assayedfor total cellular thiols. The results are expressed as a percentage of the total cellular thiols in untreated PBMC (approximately 3200 amol/cell).

had no effect. When the experiment was performed in media plus serum, NEM decreased the total cellular thiols by 42% and blocked PHA responsiveness by 69%. NHM, however, failed to decreasethe total cellular thiols under these conditions. The lack of inhibitory activity of NHM and maleimide cannot be attributed to weaker thiol reactivity. As can be seen in Fig. 6, both of these compounds reacted with glutathione more rapidly than did NEM. Interestingly, the inhibitory maleimide A25,338-3 also reacted with glutathione at a faster rate than did NEM. All of the maleimides, including NEM, reacted completely with cysteine and 2-ME in lessthan 10 set (data not shown).

Y a

0I 0“,

I

TIME

(min)

FIG. 6. Thiol reactivity of NBM analogs. Solutions of 1 &(A) NHM, (B) maleimide, (C) A25,338-3, and (D) NEM were treated at time 0 with 1 mM reduced glutathione. The loss of absorbance represents the reaction of the maleimide alkene with cysteine of glutathione.

MALEIMIDES

191

DELAY T-CELL RESPONSE TABLE 5

Effect of NEM on Proliferation and Viability of PHA Blasts”

NEMWf) 0

1 5 10

[‘H]TdR Uptake (cpm)

% Viability

65,038 + 3043 33,447 f 13157 572-c 347 IOO? 25

90.7 79.3 4.8 0

’ Cells were plated at 5 X IO5cells/ml in media + 10% AGHS + 10% IL-2 in the presence of NEM. [3H]TdR was added at time = 0 and harvested 24 hr later. Viability was determined by washing the cells once in HBSS and resuspending them in 0.2% trypan blue dye. Viable and dead cells were enumerated on a hemacytometer. The data are expressedas a percentage of the control.

Efict of NEA4 on PHA blasts. Although NEM was found to have no inhibitory activity when added after PHA, the known antimitotic activity of NEM suggested that it should be capable of blocking T-cell proliferation. The addition of NEM immediately prior to pulsing the PHA cultures with [3H]TdR at 72 hr also failed to block uptake of the [3H]TdR (data not shown). However, the cells are not actually dividing at this time. To determine the effect of NEM on proliferating cells, PHA blasts were isolated from 3-day-old cultures and grown for 6 days in media containing IL-2. Unlike unstimulated cells, PHA blasts were killed by 5-10 PM NEM, and even 1 PM NEM killed >20% of the cells (Table 5). DISCUSSION The studies presented here demonstrate the usefulness of NEM and other maleimides as probes for studying early events in human T-cell activation. Nonpolar maleimides (NEM, NPM, A25,335-9, A25,337-5, and A25,338-3) appear to block very early events in the activation process,as shown by the fact that they had to be present at T = 0 in order to be fully inhibitory. Similar results were observed in the MLC, where complete inhibition by NEM was observed only when NEM preceded the addition of allogeneic cells. These results contradict the findings of Chaplin and Wedner, who reported that NEM was inhibitory when added late in the culture (25). It is possible that the medium used in their experiments (Medium 199 supplemented with 15% fetal bovine serum), which contains a lower concentration of thiols than the medium we used, allowed more NEM to enter the cells and thereby produced inhibition at later times. However, we noted that 20 pA4 NEM was not inhibitory when added to the PHA assayafter 1 hr. The mechanism by which NEM blocks T-cell activation is thiol specific. The inhibitory activity of NEM was abolished by pretreatment with equimolar concentrations of 2-ME. Excess 2-ME (50 pLM) blocked the inhibitory effect of 10 PALM NEM when the two compounds were added simultaneously to the cultures, but 2-ME had no effect when added only minutes after NEM. 2-ME reacts with NEM stoichiometritally in lessthan 10 set and would therefore be expected to inactivate any unreacted NEM still present in the culture at that time. The fact that 2-ME has no effect when added 10 min after NEM demonstrates the rate at which NEM reacts with the cells. However, it should be noted that PBMC pretreated with NEM for 30 min and then

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washed were not as inhibited as cells to which NEM was added at the time ofstimulation. Thus, the reaction between NEM and the cellular suhhydryls regulating T-cell activation may actually proceed rather slowly, but unreacted NEM is not accessible to 2-ME after the first few minutes. On the other hand, NEM may simply be more inhibitory if it is available when the cells are being activated. It has been suggestedthat NEM blocks lymphocyte proliferation by disrupting microtubules (20,27), but as yet there is little data regarding other sullhydryl-containing structures in the lymphocyte that may also be impaired by NEM. It does not seem likely that NEM inhibits T-cell proliferation by reacting with exofacial sulfhydryls. Pretreatment of PBMC with 100 PM BESA, a membrane-impermeable sullhydrylalkylating agent, had no effect on the proliferative response (data not shown). Similarly, treatment of PBMC with the polar maleimides NHM and showdomycin did not result in significant inhibition. Furthermore, when PBMC were treated with NHM in the presence of serum, no decreasein the level of total cellular thiols could be detected. These data suggestthat the sulfhydryls critical for human T-cell activation are intracellular. In contrast, mm-me T cells appear to have exofacial membrane sullhydryls that are also involved in the activation process (36,37). The fact that NEM inhibited T-cell proliferative responsesonly when added prior to activation of the cells suggeststhat under these experimental conditions, NEM may be functioning through reactions with a limited number of cellular structures. In support of this hypothesis, NHM was found to decreasethe total cellular thiols by 38% without inhibiting the proliferative response. NEM reduced the total cellular thiols by 50%, but inhibited proliferation by 99% (Fig. 5). NEM therefore reacts with a small percentage of cellular thiols (12%) that do not react with NHM, and these thiols appear to be critical for T-cell activation. Since NEM in the presence of serum blocked only 42% of the cellular thiols and still inhibited T-cell activation by 69%, as few as 4% of the total cellular thiols may be critical for T-cell activation. The fact that the maleimide probe had to be nonpolar in order to inhibit T-cell activation suggests that the critical thiols may exist in nonpolar regions of the proteins. This mechanism of action of nonpolar maleimides has been demonstrated with various enzymes (22, 38). Although the nonpolar maleimides were observed to block early activation events, the effect was spontaneously reversed. Since the maleimide thiol ether linkage is resistant to acid hydrolysis (39), it is unlikely that the maleimide is cleaved from the protein to which it is bound. More likely, the protein is synthesized de IZOVO, after which the activation process can proceed. However, the 48- to 72-hr delay in the MLC proliferative response did not affect the generation of CTL. NEM appears likely to inhibit T-cell activation at least in part by blocking tubulin polymerization. Since microtubule polymerization is required for both agglutination and mitogenesis (40, 41), and cytoskeletal-associated changes precede the earliest changesassociatedwith cell replication (42,43), new tubulin might have to be synthesized after NEM treatment before the cells can agglutinate and proliferate. It is possible that spontaneous reversal of NEM inhibition after 48 hr was the result of the synthesis of new tubulin. When sufficient tubulin is present, the polymerization of MT may occur and the cells would begin to agglutinate. In agreement with Pfeifer and Irons (20) we observed a direct correlation between inhibition of PHA-induced agglutination and inhibition of [3H]TdR uptake by NEM treatment. More important, the spontaneous reversal of NEM inhibition was characterized by the resumption of agglutination.

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Although the above data argue strongly in favor of NEM inhibiting T-cell activation by interfering with MT, it is possible that NEM inhibits T-cell activation by alkylating proteins other than, or in addition to, tubulin. For example, it has recently been postulated that the Gi component of adenylate cyclase may be involved in the binding of phospholipase C to the cell membrane (44). Gi has a GTP-binding site that can be alkylated by NEM, but only when the protein has been activated by an agonist (45) suggesting that the critical sulthydryl group is exposed only during the activation process.Alkylation of Gi by NEM might explain why NEM is more inhibitory when added at T = 0 than when the cells are pretreated and washed. Thus, it is possible that other proteins are affected by NEM and their inactivation contributes to the delay in T-cell activation. It appears that the lymphocytes exhibit varying degreesof sensitivity to NEM at different stagesof the cell cycle. We noted that 10 PMNEM blocked the proliferative response of lymphocytes to mitogens and alloantigens when added at time = 0 (presumably G,), but had little inhibitory effect when added to PHA or MLC assays0.2518 hr after initiating the cultures (G, , S, and G2). However, when we took 3-day-old PHA blasts and cultured them in the presence of IL-2 for 6 days, the addition of 5-10 PLMNEM not only halted [3H]TdR uptake, but resulted in greater than 95% cytotoxicity within 24 hr (Table 5). The mitosis of PHA blasts is MT dependent, and interruption of the MTs during mitosis may account for the highly toxic effect seen in these cultures. The PHA blasts were observed to swell and lyse within 2 to 4 hr after being treated with NEM, suggesting that NEM can affect osmoregulation in lymphocytes undergoing mitosis but not in lymphocytes that are quiescent. Thus, NEM appears to delay activation of lymphocytes in Go and kill lymphocytes in M, but it has no noticeable effect on lymphocytes in G, , S, or GZ. These experiments demonstrate that cellular thiols have different sensitivities to various maleimide probes. It is apparent that the critical thiols involved in the early events in T-cell activation are only reactive with nonpolar maleimides. The data suggestthat only 4% of the 3200 amol of thiols/cell(32) may be critical for various aspects of the activation process.Experiments are now in progressto identify specific cellular thiols that regulate T-cell activation. REFERENCES 1. Rothstein, A., Curr. Top. Membr. Tramp. 1, 135, 1970. 2. Boumendil-Podevin, E. F., and Podevin, R. A., Biochim. Biophys. Acta 467,364, 1977. 3. Hare, J. D., Arch. Biochem. Biophys. 170,347, 1975. 4. Holmgren, A., Biochem. Sot. Trans. 5,611, 1977. 5. Vallee, B. L., Ulmer, D. D., Annu. Rev. Biochem. 41,91, 1972. 6. Goldberger, R. F., Epstein, C. J., and Anfmson, C. B., J. Biol. Chem. 238,628, 1963. 7. Askonas, B. A., and Parkhouse, R. M. E., Biochem. J. 123,629, 197 1. 8. DeMeyts, P., Methods Recept. Res. 9,30 1, 1976. 9. De Maeyer, E., and De Maeyer-Guignard, J., Ann. N. Y. Acad. Sci. 350, 1, 1980. 10. Loor, F., Adv. Immunol. 30, 1, 1980. 11. Oliver, J. M., Albertini, D. F., and Berlin, R. D., J. Cell. Biol. 71,921, 1976. 12. Amy, C. M., andRebhun, L. I., J. CellPhysiol. 100, 187, 1979. 13. Kwock, L., Wallach, D. F. H., and Hefter, K., Biochim. Biophys. Acta 419,93, 1976. 14. Schoot, B. M., Van Ernst-De Vries, S. E., Van Haard, P. M. M., De Pont, J. J. H. H. M., and Bonting, S. L., Biochim. Biophys. Acta602, 144, 1980. 15. Anderson, R. E., and Warner, N. L., Adv. Immunol. 24,215, 1976. 16. Kendall, P. A., and Hutchins, D., Zmmunology35, 189, 1978. 17. Hurvitz, D., and Hirschhorn, K., N. Engl. J. Med. 273,23, 1965.

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