Modulation of T-cell function

CELLULAR

IMMUNOLOGY

a,453469

(1981)

Modulation

of T-Cell Function

II. Chemical Basis for the Involvement of Cell Surface Thiol-Reactive Sites in Control of T-Cell Proliferation’ RANDOLPH J. NOELLE AND DAVID A. LAWRENCE Department of Microbiology

and Immunology, Albany Medical College of Union University, Albany, New York 12208

Received August 18, 1980; accepted December 17, 1980 Chemical oxidation or reduction of lymphocyte cell surface thiol or disulfide groups, respectively, has been shown to alter the proliferative activity of murine T cells. S-2-(3-aminopropylamino)ethylphosphothioic acid, a compound containing no free thiol group until it is intracellularly dephosphorylated. did not enhance Con A-inducedproliferationwhichsuggestedthat thiols did not mediate proliferative enhancement via an intracellular mechanism. Glutathione, an impermeant thiol, enhanced T-cell proliferation 68% as effectively as 2-mercaptoethanol (2-ME), which suggested that the thiol-sensitive site was at the cell surface. A battery of structural analogs to 2-ME was employed to elucidate the chemical requirements for the biological activity of the thiols. The necessity for a hydrogen-binding moiety on the thiol reagent was determined by the use of non-hydrogen-binding analogs and by competitive inhibition of the thiol-enhancing activity of 2-ME by non-thiol-containing hydrogen-binding analogs. Pretreatment of cells with the copper:phenanthroline complex (CUP), an impermeant oxidant of thiol groups, reduced the Con A-induced response >79%; however, the presence of 2-ME in culture completely reversed the inhibitory effect of CUP pretreatment. Oxidation of T cells by high oxygen tension (17% 0,) also ablated the Con A response but did not alter the response to Con A + 2-ME. Protection from oxidative inhibition also was afforded T cells by sequential reduction and blockage of cell surface thiol groups. Finally, a model which correlates the chemical study of cell surface residues with T-lymphocyte responsiveness is presented.

INTRODUCTION Cell surface elements of murine T cells have been investigated both antigenically (1, 2) and chemically (3, 4) in an attempt to define their involvement in T-cell transformation. The functions of cell surface carbohydrates in the initiation of lymphocyte proliferation have been extensively studied by utilizing plant lectins which bind to specific saccharide residues and induce mitosis (5). Cell surface sialoglycoproteins have been implicated in the activation of T lymphocytes by studies illustrating that the formation of carbohydrate-derived aldehyde groups, on the cell surface, trigger lymphocyte mitosis (4). It has been suggested that the aldehyde groups interact with other functional moieties on the cell surface producing chem’ This work was supported by Public Health Service Grant AI 12527 from the National Institute of Allergy and Infectious Diseases. 453 0008-874918 l/080453-17$02.00/O Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form rcscrvcd.

454

NOELLE

AND LAWRENCE

ical crosslinks and aggregations of specific membrane constituents. In contrast, crosslinkages between other cell surface functional groups, such as amino groups, have been shown to be ineffective in inducing transformation (6). These studies indicated that chemical alterations of particular cell surface functional groups can result in the activation of specific lymphocyte activities. Interest in cell surface thiol (SH) and disulfide (S-S) groups has developed based on the findings that 2-mercaptoethanol (2-ME)-treated’ T cells (in the absence of serum) displayed heightened proliferative responses to concanavalin A (Con A) (7). 2-ME was shown to enhance the response of unselected and Ly-l+ T-cells to Con A (sevenfold) and was shown to be a strict requirement for Con Ainduced proliferation of macrophage-depleted preparations of Ly-2,3+ cells. 2-MEenhanced T-cell proliferation was shown to be due to a direct effect on T cells and not mediated by B cells or other accessory cells (7). The purpose of this study was to investigate whether the T-cell-associated thiol groups involved in enhancing proliferative activity were located within the cell or associated with the plasma membrane. Previous reports have shown that alterations in lymphocyte thiol groups depressed adrenalin responsiveness of rat lymphocytes (8), reduced graft vs host reactions (9), modified amino acid transport in mouse thymocytes (lo), modulated guanyl cyclase activity in pig lymphocytes (1 1), affected T-cell-mediated cytolysis ( 12), and induced morphological changes of lymphocyte plasma membranes (13). In addition, preliminary reports from our laboratory (14, 15) have indicated that T-cell surface thiol oxidation and thiol blockage can alter in vitro helper activity to sheep red blood cells (SRBC) and differentially affect the activities of Ly-defined subpopulations of T lymphocytes. The experiments reported herein examine the nature of the thiol-reactive site that is responsible for altered T-cell activities. A model based on the results of the experiments reported in this paper has been presented which suggests that the status of the thiol groups on the cell surface can govern T-cell responsiveness. MATERIALS

AND

METHODS

Animals. Female CBA/J mice (Jackson Laboratory, Bar Harbor, Maine) 2-4 months of age were used for all experimental cell preparations in these studies. Mice were maintained on laboratory chow and acidified water, pH 3.0, ad libitum. Ceil preparations. Spleen cells were aseptically removed from the mice after exsanguination. Spleen cells were teased and allowed to settle to obtain single-cell suspensions as previously described (7). T-Cell preparations used in these studies were fractionated by (a) glass wool and nylon fiber’columns (7), or (b) affinity purification using purified rabbit anti-mouse Fab-coated petri dishes as described by Wysocki and Sato ( 16). B cells were isolated from spleen cell suspensions after ’ Abbreviations used: 2-ME, 2-mercaptoethanol; MESA, mercaptoethanesulfonic acid; GSH, reduced glutathione; BESA, 2-bromoethanesulfonic acid; BPSA, 2-Bromopropanesulfonic acid; NEM. N-ethylmaleimide; dBBr, dibromobimane (Thiolyte); qBBr, guar.-aminobromobimane (Thiolyte Monoquat); CUP, copper:phenanthroline; DMD, diamide; WR2721, S-2-(3-aminopropylamino)ethylphosphothioic acid, Con A, concanavalin A; BSS, balanced salt solution; [3H]TdR, [‘Hlthymidine; E.C., enhancement capacity; SRBC. sheep red blood cells; LETS, large extracellular transformation-sensitive protein; ZOGS, surface-acting asymmetrical disultides.

INVOLVEMENT

OF T-CELL

SURFACE

THIOLS

IN PROLIFERATION

455

anti-Thy- 1.2 + complement treatment, as described previously (7). For estimating intracellular levels of reduced glutathione, red cells were first removed from spleen cell preparations by the following procedure. Red cells were first hypotonically lysed by treatment with 0.83% ammonium chloride, then removed from the spleen cell suspension by density gradient centrifugation with Ficoll-Paque (Pharmacia, Piscataway, N.J.) at 500g for 10 min at 4°C. The cells at the interface were harvested, washed twice, and further fractionated into B-cell-depleted preparations by affinity adherence, as described above. Cultures. The culturing techniques employed were described by Mishell and Dutton (17) with the following modifications. Medium contained 5 X 10m5 M 2ME or other thiols, where indicated. For the proliferative studies, cells were cultured at 2 X lo5 cells/microwe11/200 ~1 with 2 pg/ml of Con A. Reagents. Thiols, disulfides, and blocking agents were purchased from Sigma Chemical Company (St. Louis, MO.), Wateree Chemical Company (Lugoff, S.C.), Aldrich Chemical Company (Milwaukee, Wise.), Calbiochem-Behring (La Jolla, Calif.), or Pierce Chemical Company (Rockford, Ill.) and were of the highest purity available. Recrystallized concanavalin A (Con A, Type IV; Sigma) was prepared daily at a concentration of 1 mg/ml in balanced salt solution and diluted for culture. Measurement of [‘H]thymidine incorporation. Cultures were pulsed for 6 hr with 0.5 PCi of [3H]TdR (5.7 Ci/mmol; New England Nuclear Corp., Boston, Mass.) on the day specified, harvested with a multiple cell culture processor (Skatron AS, Leibgen, Norway), and counted as previously described (7). All results presented are the mean of triplicate cultures and expressed as counts per minute per culture (cpm/culture). The results were considered significantly different when P < 0.01. Calculation of thiol enhancement capacity (E.C.). Since the absolute enhancement of the Con A response by thiols varied between experiments, the relative enhancement capacity of thiols compared to 2-ME was determined by the formula: E c = (cpm/culture; . . (cpm/culture;

Con A + thiol) - (cpm/culture; Con A + 2-ME) - (cpm/culture;

Con A) Con A) ’ ‘O”.

An E.C. value of ~100 indicated that the thiol reagent was less effective than 2ME in enhancing the Con A response (a value of <5 indicating an inactive thiol). A value of greater than or equal to 100 indicated that the reagent was equal to or better than 2-ME in enhancing Con A responsiveness. Determination of T-cell-reduced glutathione. A micromethod for measurement of reduced glutathione has been developed in our laboratory.3 Briefly, untreated or treated lymphocytes were washed twice and 10 ~1 of the cell suspension (2.5 X lO’/ml) was mixed with 15 ~1 of N-[3H] ethylmaleimide ([3H]NEM, 692 &i/ mmol; New England Nuclear) in 0.66% NP-40 (Particle Data Laboratories, Ltd., Elmhurst, Ill.) and allowed to react for 1 hr at 22°C. The mixture was deproteinized with 5 ~1 of 60% trichloroacetic acid. The sample was spun for 10 min at lO,OOOg, 5 ~1 of the supernatant spotted on cellulose MN 300 thin-layer chromatography (TLC) sheets, and run in a solvent system described by States and Segal (18) for the separation of glutathione from its adduct with NEM. Each lane of the TLC ’ Noelle, R. J., and Lawrence, D. A., Correlation of the reduced glutathione content of lymphocytes with the ability of thiols to modulate their proliferation. (Manuscript in preparation.)

NOELLE

456

AND LAWRENCE TABLE

1

Chemical Requirements of Thiols for Enhanced Concanavalin A-Induced Proliferation”

Reagent

Optimal dose (M)”

E.C.’

5 x 10-S 5 x lo-’

100 30

. .S)2

5 x 10-5

145

.SH

5 x 1o-6

52

5 x lo-’

56

Chemical structure

Mercaptoethanol Methyl mercaptoethanol

CH2CHI.. . .SH CH*CHr . . . .SH

HO....

HO....

I (3-h 2-Hydroxypropyl disulfide

(HO. . . .CHCHr..

I CK a-Thioglycerol

HO.. .CHCH2..

Dithiothreitol HO.. .CHCHr..

.SH

SO;...CH$JHr..SH

-

Cysteamine

NH 2.. .CH2CH2.. .SH

lo-’

87

Cystamine

(NHZ.. .CH2CH2.. -S-)2

5 x 10-S

61

NH *. . .CHCH*. . .SH

10-l

54

lo-)

56

5 x lo-’

64

Mercaptoethanesulfonic

acid

Cysteine

1 (56)d

-0oc Glutathione

glu-NH?. . . .CHCH*. . .SH gly-60

3-Mercaptopropionic

acid

2-Mercaptopropionic

acid

-0OC..

. .CHrCHr.. CH,CH..

. .SH

-

1

-

1

. . .SH

-

I

.SH

lo-’

202

(41)

.SH

-0oc Mercaptopropionylglycine

Gly . . . . CH,...CH....SH

I ‘X Thiolacetic acid Ethanethiol

CH,.CO.. CH,CHz..

1-Propanethiol

CH ,. . .CH#ZH>. . .SH

lo-’

190

(30)

2-Propanethiol

CH,CH . . . SH

1o-’

209

(32)

10-1

169

CH, Butanethiol

CH,CH*. . .CH2CHr..

.SH

Heptanethiol

CHI(CH&.

. .CHICH2. .SH

lo-’

59

Octanethiol

CHa(CH&.

. .CHICHI..

.SH

10-r

51

Decanethiol

CH,(CHs),.

. .CHICH2..

.SH

lo-’

42

INVOLVEMENT

OF T-CELL

SURFACE

TABLE

THIOLS

IN PROLIFERATION

457

1-Conrinued

Chemical Requirements of Thiols for Enhanced Concanavalin A-Induced Proliferation”

Reagent Cysteine sultinic acid

S-2-(3-aminopropylamino) ethylphosphorothioic acid hydrate

Chemical structure

Optimal dose (M)”

NH *. . . . .CH2CH2.. .SO,H I -0Oc HzNCH$JH#ZH~NHCH~CH~S-P0,H2. XH20

E.C.’ 1

-

I

’ Spleen cells (2 X lO’/well 200 ~1) were pulsed with 0.5 &i [‘H]TdR from 42 to 48 hr postinitiation of culture. hAll reagents were tested at final culture concentrations ranging from 5 X IO-’ to IO-’ M. The concentration eliciting the highest E.C. is listed. The results are representative of three separate experiments. Each reagent at various concentrations was tested in triplicate and variance was less than 15%. ’ E.C., enhancement capacity, calculated as described under Materials and Methods. The value rep resents the mean for the separate experiments and the variance was less than 15%. d For comparative purposes, the E.C. of selected reagents at 5 X 10m5M has also been presented.

was cut into l-cm sections and counted by liquid scintillation spectrometry. The sum disintegrations per minute from sections corresponding to the GSH-NEM adduct were utilized to estimate the amount of GSH in the sample. Control preparations of murine T cells contained approximately 3.6 X lo-l6 mol GSH/cell. RESULTS Structural Specificity of Thiols and Disul’des for the Enhancement of Con AInduced Proliferation

The enhancement capacity of 2-ME analogs, as seen in Table 1, was the relative enhancement capacity of the reagent compared to 2-ME (calculated as described under Materials and Methods). All reagents were tested at final culture concentrations from 5 X lo-’ to 1 X 10m3 M. The substitution of the hydroxyl group of 2-ME with other functional groups altered the activity of the molecule. Substitution of the secondary hydroxyl group of 2-ME decreased its effectiveness in the following order: hydroxyl(2-ME, 5 X lo-’ M) >amino (cysteamine; 5 X lo-’ M) >carboxyl (3-mercaptopropionic acid; 5 X lo-’ M) $-hydrogen (ethane thiol; 5 X 10m5 M) >methyl (l-propane thiol; 5 X lo-’ M) =-amino and carboxyl (cysteine; 10e3 M) >sulfonic acid (mercaptoethanesulfonic acid; inactive). To further examine the involvement of hydrogen bonding in 2-ME-induced enhancement of mitogen responsiveness, reagents potentially able to compete for a hydrogen-bonding site were added to spleen cell cultures containing Con A or Con A + 2-ME. As seen in Table 2, the hydrogen-bonding analogs had minimal negative effects on Con A-induced responses, but produced from 27% (ethylene glycol)-44% (ethanol) inhibition of the Con A + 2-ME response. Ethylenediamine, which contains no hydroxyl groups, also depressed the Con A + 2-ME response. This was not unexpected since an amino group also can be involved in hydrogen bonding, and mercaptoethylamine (cysteamine) also enhanced the Con A response (Table 1). The data suggest that

NOELLE

458

AND LAWRENCE TABLE

2

Inhibition of Mercaptoethanol Enhancement of Con A Responsiveness by Non-Thiol-Containing Structural Analogs” Percentage control response’ Reagent* -

Ethylene glycol Glycerol Ethanol Ethylenediamine Thiodiglycol Ethionic Acid

Con A

Con A + 2-ME

100 108 91

100

114 103 105

100

13 64 56 70 57 69

“Spleen cell cultures were pulsed from 66 to 72 hr postinitiation of culture with [‘H]TdR and the results are expressed as percentage of the Con A or Con A + 2-ME response. ‘The nonthiol, hydrogen-bonding reagents were added to the spleen cell cultures at initiation and their final concentrations were 10m4M. ’ The results represent the mean of two separate experiments. Each mean was calculated from triplicate cultures and the variance was consistently less than 15%. The Con A + 2-ME:Con A response ratios were 1.9 and 3.6.

the hydrogen-bonding capability of the hydroxyl group of 2-ME does play a role in its biological activities. The hydrocarbon spacing group also demonstrated rigid structural requirements for thiol-mediated enhancement of Con A-induced proliferation. Substitution of the primary carbon with a methyl group (methylmercaptoethanol; 5 X lO-4 M) produced a 70% decrease in the enhancing capabilities of the molecule. In contrast, substitution of the secondary carbon with a methyl group (2-hydroxylpropyldisulfide) had little consequence on the activity of the reagent. Carboxyl (2-mercaptopropionic acid) or carbonyl (thiolacetic acid) substitutions on the primary carbon rendered the reagent inactive, in comparison to the unsubstituted parent-alkane thiol analog (ethane thiol). It is important to note that only monothioglycerol was active at concentrations lower than 2-ME. This activity at micromolar concentrations apparently was due to the hydroxyl group of the tertiary carbon, since methylmercaptoethanol was substantially less effective than monothioglycerol even at loo-fold higher concentrations. The enhancing capacity of the alkane thiol family of reagents was tested using a series of reagents from ethane thiol to decane thiol. As seen in Table 1, both ethane thiol and propane thiol were effective only at millimolar concentrations. The disulfide (cystamine) of an active thiol (cysteamine) also was active. In addition, 2-hydroxylpropyl disulfide, another disulfide, was active. No reagents with substituted thiol groups were effective in enhancing Con A-induced responsiveness, e.g., cysteine sulfinic acid, thiodiglycol, and WR2721. Intracellular

and Extracellular

Thiols

In order to determine whether thiol reagents act at the cell surface or within the cell, reagents were chosen which were cell impermeant or could deliver thiol groups only to the cell interior. As seen in Fig. 1, glutathione (GSH), a membrane-im-

INVOLVEMENT

OF T-CELL

SURFACE

THIOLS

IN PROLIFERATION

459

0

60 t

Log

Ccmxntratim

(M)

I. Effect of intracellular and extracellular thiols on Con A-induced proliferation. Spleen cells were placed in culture with various concentrations of cysteine (A), glutathione (m), or WR2721 (0). The Con A response in the presence of 5 X 10d5 M 2-ME is also reported (0). All cultures were pulsed with 0.5 PCi of [‘H]TdR from 42 to 48 hr postinitiation of culture. Each point represents the arithmetic mean of triplicate cultures. Variance was less than 10%. FIG.

permeant, thiol-containing tripeptide, enhanced Con A-induced responsiveness 68% as effectively as 2-ME. Similar dose-response characteristics were seen with Lcysteine. The Con A response of purified T cells (35,4 10 +- 68 18) also was enhanced 3.6-fold (129,55 1 f 12,046) when 1 mM GSH was present in culture. This reaffirmed the lack of B-cell involvement (7) in the thiol-enhancement of Con Ainduced T-cell proliferation by GSH. S-2-( 3-aminopropylamino)ethylphosphothionic acid (WR2721; obtained from Walter Reed Army Institute of Research, Washington, D.C.) is a cysteamine analog with a phosphorylated sulfur group, which has been reported to be intracellularly dephosphorylated, liberating free thiol groups within the cell (19, 20). WR2721 was used as an indicator of whether the delivery of free thiol groups exclusively within the cell was sufficient to promote Con Ainduced proliferation. Since WR272 1 was ineffective (Fig. 1) and GSH was effective in enhancing Con A-induced transformation, cell surface thiol-mediated enhancement of T-cell proliferation was suspected. Oxidation of Cell-Associated Thiol Groups by High Oxygen Tension and Protection by 2-ME Mishell and Dutton (17) have previously reported that the in vitro primary immune response of murine spleen cells was diminished at oxygen tensions >7%. Others have reported murine primary immune responses in vitro at 17% O2 tensions (21); however, the culture medium was supplemented with 2-ME in these studies. To test whether 2-ME could protect murine spleen cells from atmospheric oxidative inhibition, spleen cells were cultured with Con A f 2-ME at low (7%) and high

460

NOELLE

AND LAWRENCE

Doy of culture

FIG. 2. Oxidation of cell-associated thiol groups by high Or tension and protection by 2-ME. Spleen cells were prepared as described under Materials and Methods, placed in culture under a 7% (0, n ) or 17% (0, q ) oxygen atmosphere with Con A (0,O) or Con A + 2-ME (a. 0). All cultures were pulsed with 0.5 PCi of [‘H]TdR for 6 hr on Days 1-5. Each point represents the arithmetic mean of triplicate cultures. Variance was less than 10%.

( 17%) oxygen tensions, and their proliferative activity was assessed. As seen in Fig. 2, spleen cells responded poorly to Con A at high O2 tensions unless 2-ME was present. Comparable results also were obtained with purified preparations of T cells (Table 3). Catalytic Oxidation of Cell Surface Thiol Groups by the Copperphenanthroline Complex Enhancement of Con A-induced T-cell proliferation by GSH suggested that alterations in the thiol groups on the lymphocyte surface were responsible for enhanced T-cell proliferative activity. Due to the sensitivity of murine T cells to high O2 tension, it was suspected that the formation of cell surface disulfides would depress Con A-induced T cell proliferation. Copper:phenanthroline (CUP) catalyzes TABLE

3

Inhibitory Effects of High O2 Tension on T-Cell Responsiveness and Reversal with 2-ME [‘H]TdR Culture conditions“

incorporation (cpm/culture)b

Con A

Con A + 2-ME

7% o*

64,810 + 4,547

142,530 f 3.180

17% o*

1,500 + 1,004

171,725 f 6,073

’ T cells were placed in culture under the oxygen tensions listed for the entire 72-br period. b T-Cell cultures were pulsed from 66 to 72 hr postinitiation of culture with 0.5 pCi [‘H]TdR the results are expressed as the arithmetic mean of triplicate cultures f SD.

and

INVOLVEMENT

OF T-CELL

SURFACE TABLE

THIOLS

IN PROLIFERATION

461

4

Chemical Oxidation of Cell Surface Thiol Groups“ [‘H]TdR Pretreatmentb

5 x lo-6 MCUP

Incorporation

(cpm/culture)’

2-ME

Con A

Con A + 2-ME

10,658 + 336

39,047 f 3,573

98,949 + 3,335

1,473 f 163

8,232 iz 1,647

96,765 f 4,752

2.5 x IO-’ M CUP

458 f 83

2,426 + 418

65,328 f 3,235

5.0 x lo+ M CUP

101 f 12

112 + 13

12,165 f 621

1 x 1o-4 M CUP

128 + 9

119 f 7

2 x 1O-4 M DMD

1,663 + 88

5,105 k 685

104,369 k 7,491

84 f 6

7,766 + 413

2 x 1O-3 M DMD

92 f 6

7,574 + 1,890

’ Spleen cell cultures were pulsed from 66 to 72 hr postinitiation of culture with [3H]TdR and the results are expressed as the arithmetic mean of triplicate cultures -t SD. * Spleen cells were treated with various concentrations of copper phenanthroline (CUP) or diamide (DMD) in BSS at a cell concentration of 1 X 10’ cells/ml for 20 min at room temperature. The cells were washed twice with BSS and placed in culture with Z-ME (5 x IO-’ M), Con A (2 rg/ml), or Con A + 2-ME. ’ Response in the absence of all mitogens was 105 cpm/culture.

the oxidation of thiol groups (22). Since CUP has been suggested to be impermeant ((23) Table 5) it was used to investigate the effects of cell surface thiol oxidation on lymphocyte proliferation. Spleen cells were preincubated in graded doses of CUP, washed free of the reagent, placed in culture and the response to Con A and Con A + 2-ME was ascertained. When cells were preincubated in 5 PM CUP, the Con A response was depressed 79% but the Con A + 2-ME response was unchanged (Table 4). When cells were preincubated in concentrations of CUP greater than or equal to the culture concentration of 2-ME, the Con A response was reduced greater than 95%, and the Con A response was unable to be restored by 5 X 10m5 M 2-ME. None of the CUP concentrations used for preincubating cells were immediately toxic to the cells as judged by trypan blue exclusion. Diamide (DMD), another thiol oxidant (24), produced similar effects on Con A +/- 2-ME-induced proliferation at concentrations 40- to 60-fold greater than those of CUP. These data indicate that murine spleen cells are sensitive to thiol oxidation and that the sensitive thiols are on the cell surface. Furthermore, unlike the Con A + 2-ME response, pretreatment of lymphocytes with CUP and DMD irreversibly inhibited the response of the spleen cells to 2-ME, which has been shown to be due to B-cell proliferation (7, 25). These differential inhibitory effects of oxidants on the Con A + 2-ME response versus the 2-ME response again indicate that two independent phenomena are involved (7) and suggests that B cells are more sensitive to surface oxidation than T cells (5 X 10e6 M CUP; Table 4). Cell Surface Thiol Reduction, Blockage, and Oxidation

In the previous sections, the results indicated that thiols enhanced lymphocyte transformation by either (a) disulfide exchange and/or (b) preventing membrane

462

NOELLE

AND LAWRENCE

WR272l GSH

2-ME

FIG. 3. Cell surface thiol reduction, blockage, and oxidation. T cells were pretreated with BSS (open bars) or BSS containing I X IO-’ M 2-ME (slashed bars) for 45 min at 37°C washed twice, and placed in cultures containing Con A (2 fig/ml) and one of the following thiol reagents: BESA (1 X 10e5 M); BPSA (1 X IO-’ M); MESA (1 X lo-;3 M); GHS (1 X 10-j A4); 2-ME (5 X IO-’ M); or WR2721 ( 10-4-10-6 M). The cells were placed in a 17% 01 atmosphere and pulsed with [‘H]TdR from 66 to 72 hr postinitiation of culture. The results represent the arithmetic mean of triplicate cultures + SD.

disulfide bond formation by preexisting cell surface thiol groups. In order to discriminate between these two possible mechanisms of thiol action, the following studies were undertaken. T cells were first pretreated with BSS (T:S-S) or BSS plus 10e3 M 2-ME (T:SH). Pretreatment of T cells with 10e3 M 2-ME previously has been shown to enhance Con A responsiveness under 7% O2 culture conditions (7) and was employed in this study to generate T cells with more free thiol groups on their surface (T:SH). T:S-S and T:SH were washed twice and placed in culture under 17% O2 tension with the impermeant thiol blockers 2-bromoethanesulfonic acid (BESA) and 2-bromopropanesulfonic acid (BPSA), or various other thiol reagents. As seen in Fig. 3, T:S-S or T:SH were unresponsive to Con A under 17% O2 tension. If T:S-S were placed in culture with Con A plus low5 M BESA, a 3.7fold enhancement in responsiveness was observed; however, if T:SH were placed in culture with BESA, the response observed was approximately 10.2-fold higher than T cells which had not been blocked by a thiol reagent. BPSA only enhanced the responsiveness of T:SH and not T:S-S. A permeant thiol blocker (NEM) at similar concentrations completely inhibited the Con A +/- 2-ME-induced response (data not shown). Also seen in Fig. 3 is the protective capacity of lop3 M GSH and 5 X 10e5 M 2-ME. Although low3 M MESA was ineffective in enhancing Con A-induced responsiveness under 7% O2 (Table l), it was marginally effective in protecting T cells from oxygen-mediated inhibition. The phosphorylated thiol compound, WR272 1, was completely inactive. T-Ceil-Reduced Glutathione Content after Treatment with Permeant and Impermeant Thiol Reagents The concept that cell surface thiol groups are important in modulating T-cell responsiveness was based on the assumption that charged thiol reagents (BESA)

INVOLVEMENT

OF T-CELL

SURFACE THIOLS TABLE

T-Cell-Reduced

Pretreatmentb (M) lo-’ 2-ME IO-’ GSH lO-4 BESA lO-5 NEM 10e4 dBBr 10m4qBBR 1.5 x lo-5 CUP lo-4 CUP 5 x lO-4 DMD

IN PROLIFERATION

463

5

Glutathione Content after Treatment with Permeant and Impermeant Thiol Reagents” IO-l6 mol GSH/cell

Percentage control’

4.5 3.4 3.7 0.1 0.3 3.6 3.6 1.4 I.3

124 95 103 4 9 100 103 39 36

a T cells were isolated, free of erythrocytes, as described under Materials and Methods. “T cells were suspended in BSS (I X IO’ cells/ml) and treated for 20 min at room temperature, washed twice, and then used for GSH estimation. ‘Percentage control was calculated as follows: GSH content of reagent-treated T cells per GSH content of BSS-treated T cells X 100. The results are representative of two to three separate experiments.

or high-molecular-weight thiol reagents (GSH and CUP) were T-cell membrane impermeant. To examine the permeant nature of the reagents on T cells, T cells were treated with the thiol reagents in the same manner as described in the previous sections and the reduced GSH content was immediately assessed by a recently developed radiometric thin-layer chromatography technique as described under Materials and Methods.3 If the content of reduced GSH in the cells was lowered by reagent treatment, the reagent was able to either block (GSH + GSX) or oxidize (GSH + GSH + GSSG) intracellular reduced GSH, which suggests that the reagent was able to enter the cytosol of the cell. If no change in the GSH content was apparent after treatment, then the reagent was unable to penetrate the cell membrane. Table 5 indicated that thiol-blocking reagents carrying a strong negative (MESA and BESA) or positive (qBBr) charge could not alter intracellular GSH concentrations. Uncharged thiol blocking agents, dBBr and NEM, lowered the available reduced GSH 9 1% and 96%, respectively. 2-ME ( 1 X 10m3M) increased the intracellular content of reduced GSH; whereas, reduced GSH (1 X 10m3 M) had no effect on intracellular reduced GSH levels. CUP, at low concentrations ( 1.5 X lo-’ M) did not diminish the intracellular content of reduced GSH; however, 5 X 10V6 M CUP produced 79% inhibition of Con A-induced proliferation (Table 4). CUP at higher concentrations ( 10V4 M) depressed reduced GSH levels >60%, as did DMD, but 10e4 M CUP also reduced cell viability. DISCUSSION The manner by which simple sulfhydryl compounds, like 2-ME, promote the growth (26) or activities (7-l 2) of murine lymphoid cells in culture has been studied herein. The results from this work indicated that the chemical status of the thiol groups on the T-cell plasma membrane governed the activities of the cells, i.e., oxidation of T-cell surface thiol groups inhibited Con A-induced T-cell activation.

464

NOELLE

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Broome and Jeng (26) have previously reported the growth-promoting capacity of many thiol compounds for murine neoplastic lymphoid cells. The biological activities of these thiols, as well as others, have been reexamined (a) to ensure that the thiol reagents produce similar effects on normal T-cells; (b) to better quantitate their relative enhancing activities on murine T-cell responsiveness to Con A; (c) as a tool to elucidate the nature of the thiol-sensitive site; and (d) to aid in the development of a model to explain the phenomenon of thiol-induced alterations of T-cell activities. Most of the thiols that were reported to enhance neoplastic lymphoid cell growth are shown in this report to promote murine lymphocyte transformation (Table 1). One notable exception is reduced glutathione, which at a culture concentration of 10e3 M, enhanced splenic and T-cell Con A responsiveness 2.4 and 3.7-fold, respectively. The fact that GSH was membrane impermeant (27; Table 5) indicated that there may be a site on the lymphocyte plasma membrane which is sensitive to thiol modulation. If the assumption is made that all of the thiols enhanced T-cell proliferation by acting at the same site and in the same manner, the following statements can be suggested about the active site on the Tcell membrane. (i) Since monothioglycerol and 2-ME were effective promoters of Con A-induced T-cell proliferation at concentrations far lower than those of nonhydrogen-bonding analogs (e.g., ethane thiol), hydrogen bonding may play a role in positioning and maintaining the thiol reagent at the active site, as previously suggested (25). Cysteamine, also capable of hydrogen bonding, exemplified this point. The inhibitory capacity of hydrogen bonding analogs (Table 2) confirmed the importance of the hydroxyl group of 2-ME in the enhancement of lymphocyte proliferative activities. (ii) Since disulfides of active thiols also were active, the thiol does not function by reducing a disulfide at the active site, but probably exchanges or occupies the active site by disulfide interchange. (iii) There exists sensitive steric hindrance and charge effects near the thiol-containing active site. For example, substitution of a methyl group on the primary carbon of 2-ME (methylmercaptoethanol) diminished the activity by >70%, and the substitution of any polar groups on the primary carbon obliterated all enhancing activities. However, the secondary carbon can tolerate a variety of substitutions, including rather bulky methoxy or charged carboxyl groups. It appears that steric or charge hindrances do not play an important role if located >2-4 A from the primary thiol group. (iv) Enhancement of T-cell Con A-induced responsiveness by glutathione indicates that thiols need not penetrate the cell to be functionally active. Not all impermeant thiols, however, were active enhancers of Con A-induced responsiveness. MESA, a strong negatively charged thiol, was completely inactive. The reason why MESA was inactive and GSH was active may be due to the sulfonic acid group of MESA or perhaps the hydrogen-bonding character of GSH. Further, the inactivity of WR2721 indicated that the introduction of intracellular thiol groups had no positive effect on Con A responsiveness. The site of action of thiols, like 2-ME, for the enhancement of Con A responsiveness can be suggested to be on or within the lymphocyte plasma membrane in a hydrophobic environment close to the hydrophilic surface of the cell. Also, the activity of the thiol reagents relies on its ability to hydrogen bond. The structural thiol analogs presented in this study served as one class of probes used to reduce or exchange with the thiol-sensitive site on the plasma membrane. Another category of reagents which chemically alter free thiol groups are thiol

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oxidants such as CUP. CUP is composed of two molecules of phenanthroline and one molecule of copper (22) and the overall molecular complex has an impermeant, hydrophobic character (23). Chemical oxidation by CUP of the lypmphocyte surface catalyzes disulfide bond formation and treatment of spleen cells with CUP diminished the Con A response. As shown in Table 4, when cells were treated with 5 x 10e6M CUP or 2 X lop4 M DMD, they became >79% less responsive than untreated cells. The inhibition of mitogen responsiveness by CUP pretreatment was completely reversible when 2-ME was present in culture. These facts also support the contention that the thiol-reactive site is at the level of the plasma membrane. Culturing T lymphocytes under higher O2 tensions also lowered their capacity to respond to mitogen (Table 3). As in the CUP study, 2-ME could completely reverse the deleterious effects of 17% 0,. Using the sensitivity of T cells to 17% 02, the question of protection of mitogen responsiveness from 17% 0, inhibition was addressed. Figure 3 illustrated that when T cells were pretreated with 2-ME and cultured under 17% 02, no protection from oxidative inhibition was afforded. This pretreatment previously has been shown to enhance the Con A responsiveness of T cells cultured at 7% O2 (7). The postulated mechanism by which thiol reagents alter the Con A-induced responsiveness of T cells is illustrated in Fig. 4 and summarized as follows: (I) Elimination of Surface Disulfides (Enhances Responsiveness)

Murine lymphocytes have been reported to have a complement of free thiols (-SH) and disulfide groups (-S-S) on the cell membrane (28, 29). As previously reported (7) under 7% O2 conditions the T cells responded to Con A (Fig. 4A) and this response to Con A could be enhanced two- to sevenfold when the T cells were pretreated with 2-ME or cultured in its presence (Fig. 4B). However, pretreatment of T cells with 2-ME was not adequate for protection against 17% 0, culture conditions (Fig. 3) since the cells probably readily revert to the oxidized state which results in unresponsiveness (Fig. 4C). 2-ME and other specific thiol reagents when present in the cultures under 17% O2 could maintain T-cell responsiveness or convert them to a highly responsive state (Fig. 4B). Even MESA, which was unable to enhance the Con A response under 7% O2 conditions, was able to protect T cells from extensive surface oxidation so that the cells could show some responsiveness to Con A under 17% O2 conditions (Fig. 3). An important observation that has been reported previously (26) and confirmed in this study is that the oxidized form of active thiols also were active. Therefore, the enhancing capacity of thiols is not necessarily due to their ability to reduce protein-protein disulfide bonds on the cell surface, but possibly to interrupt protein crosslinks by disulfide exchange. Disulfides (cysteamine, 2-hydroxypropyl disulfide) are proposed herein to act by forming mixed disulfides with membrane proteins and thereby diminishing protein-protein disulfide crosslinkages on the cell surface (Fig. 4E). Therefore, enhancement of the reactivity of T cells to Con A is illustrated to be due to the elimination of membrane disulfide bonds by reduction (Fig. 4A) or disulfide exchange (Fig. 4E). (2) Thiol Oxidation

(Diminishes responsiveness).

If T cells are exposed to high oxygen tension in culture (Fig. 2) or if the lymphocyte surface thiols are oxidized by CUP (Table 4), the Con A-induced respon-

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FIG. 4. Hypothetical scheme to explain the effects of thiol reagents on T-cell responsiveness. R, responsive state; HR, high responsive state; UR, unresponsive state. For details, see Discussion.

siveness is diminished by ~80%. The diminuation in responsiveness is believed to be due to the formation of disulfide crosslinks between membrane proteins (Fig. 4C). Cell treatments that prevent or reverse cell surface disulfide bond formation maintain or augment mitogen responsiveness. (3) Blockage

(Slight

Enhancement).

Blockage of T-cell membrane thiol groups has variable effects which are dependent on the blocking reagent used. In Fig. 3, an enhancement was observed by BESA; however, BPSA as well as qBBr (data not shown) had no protective capabilities on non-pre-treated cells. Although thiol-blocking reagents have a high specific activity for thiol groups, it has been established that there exists a range of reactivity toward particular thiol groups based on the exact chemical nature of the blocking reagent (30). Therefore, the effectiveness of a thiol-blocking agent may depend upon its ability to react with specific membrane thiol groups to prevent disulfide crosslinks between certain proteins. Since thiol blockers prevent

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thiol blockers were re-

(4) Reduction/Blockage/Oxidation. By sequentially treating reactive (Fig. 4A) or unreactive (Fig. 4C) T lymphocytes with a reducing agent (Fig. 4B), then an impermeant blocking agent (Fig. 4F), the proliferative activity of the T cells can be rendered resistant to oxidative inhibitory effects (Fig. 4G). This indicated that the prevention of disulfide crosslinkages on the T-cell surface, generated by 17% O2 conditions, permitted the T cells to maintain a mitogen responsive state. If the cells were not blocked after reduction, they were still sensitive to 17% O2 conditions. Therefore, treatment of T cells with 2-ME reduces disulfide crosslinks between proteins (protein-SS-protein + protein-SH + protein-SH) and blockage (protein-SX) prohibits oxidative regeneration of protein-protein disulfide bonds. In Fig. 4, thiol reagents were proposed to interrupt disulfide bonds between membrane proteins, resulting in the alteration of the lymphocyte proliferative activities. Alterations of cellular activities by thiol-induced modification of membrane proteins has been suggested in other systems (31, 32). For example, the large extracellular transformation-sensitive protein (LETS) on the surface of hamster cells has been reported to exist as complexes which are bridged to the membrane by disulfide bonds (3 1). In addition, Hynes and Destree (32) have reported that LETS is present on the cell surface almost exclusively as disulfide-bonded complexes including homodimers or homopolymers as well as being involved in inter- and intramolecular complexes by disulfide linkages. Reduction of LETS by DTT was shown to eliminate the ability of LETS to confer flattened colony morphology and increase adhesivity when added to transformed cells. DTT treatment also caused a time and temperature-dependent release of LETS from the cell surface. Since the presence of LETS on the cell surface is correlated with cell transformation (33) and reducing agents alter the association of LETS with the plasma membrane, reducing agents may be able to modulate the transformability of cells. Other reports have considered the importance of a thiol-sensitive step in lymphocyte activation and function (9, 34-36) and have compared the macrophage requirements for T-cell activation with the redox state of the T cell necessary for activation (7, 36). Chaplin and Wedner (34) have reported that DMD added to culture inhibited mitogen-induced responses. In the same study, it was shown that thiol oxidation did not alter the amount of Con A bound to the cells. Field et al. (9) have shown that certain lymphocyte surface-acting, asymmetrical disulfides (ZOGS) could effectively block graft vs host reactions in vivo. It was suggested that ZOGS interchanged with free thiol or disulfide groups on the lymphocyte plasma membrane and depressed graft vs host reactions. Recently, it has been reported that T-cell-mediated cytolysis could be prevented if the effector cells were treated with thiol-blocking reagents (12). In addition, macrophage help for T-cell activation can be enhanced by 2-ME when a suboptimal number of macrophages is present (7) and it has been suggested that macrophage suppression may relate to macrophage production of oxidative products (36). Hence, there exist many activities of lymphocytes which have been documented to be sensitive to cell surface thiol modulation.

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The thiol-reactive reagents employed in these studies (BESA, CUP, and 2-ME) have been shown to alter T-cell membrane proteins (37). In addition, CUP, an impermeant oxidant (23; Table 5), can inhibit T-cell activation, and BESA, an impermeant thiol blocker (Table 5), can protect T cells from oxidative inhibition; however, oxidative inhibition probably is not due only to the loss of thiols at the cell surface. The surface oxidation may require the cell to utilize its cellular energy to repair the oxidative damage on the surface, thereby diverting the energies required for cell proliferation. Excessive concentrations of CUP (Table 5) did produce a decrease in the intracellular GSH levels, possibly because some of the GSH was being utilized to reduce the oxidized surface membrane proteins. Surface oxidation is known to initiate the reducing potential of a cell (27). Furthermore, oxidant injury of lymphocytes has been suggested to result from impairment in glucose metabolism which may implicate GSH metabolic pathways (38). Presently, the biochemical basis for murine lymphoid cell hypersensitivity to surface thiol oxidation is under investigation. Preliminary results indicate that the content of reduced glutathione in mouse splenic T cells is l/2 to l/3 of that found in human peripheral blood lymphocytes.’ The depressed level of reduced GSH in murine T cells may be responsible for their hypersensitivity to membrane thiol oxidation, since intracellular GSH has been suggested to maintain the thiol status of the cell membrane (27). Experiments are currently under way to test this hypothesis. ACKNOWLEDGMENTS The authors would like to thank Dr. Peter Weber (Department of Biochemistry, Albany Medical College) for his helpful advice and his critical evaluation of the data. In addition, we would like to thank Georgia Lambrinos and Kathy Smith for their technical assistance and Kathleen Cavanagh for her aid in the preparation of the manuscript.

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