Four sulfhydryl-modifying compounds cause different structural damage but similar functional damage in murine lymphocytes

Chem~-Biol~ Interactions, 68 (1988) 137--152 Elsevier Scientific Publishers Ireland Ltd.

137

FOUR SULFHYDRYI~MODIFYING COMPOUNDS CAUSE D I F F E R E N T S T R U C T U R A L DAMAGE B U T S I M I L A R FUNCTIONAL DAMAGE IN M U R I N E LYMPHOCYTES

DAVID D. DUNCAN and DAVID A. LAWRENCE*

Department of Microbiology and Immunology, Albany Medical College, Albany, N Y 12208 fU.S.AJ (Received January 14th, 1988) (Revision received May 18th, 1988) (Accepted May 19th, 1988)

SUMMARY

Four thiol-modifying compounds were used to inhibit murine lymphocyte mitogenesis. The compounds were a copper sulfate/O-phenanthroline complex (CUP) to oxidize surface thiols, N-ethyl maleimide (NEM) to alkylate surface and intracellular thiols, V,L-buthionine-S,R-sulfoximine (BSO) to prevent synthesis of glutathione, and hydrogen peroxide, which reacts with various cellular constituents, including sulfnydryls. Splenic lymphocytes were incubated with one of the four compounds, washed, and then stimulated with the B cell mitogen, LPS, or the T cell mitogen, Con A. In spite of their differing chemical reactivities and differing effects on cell viability, lipids, and total, protein, and non-protein thiols, the four sulfhydryl-modifying compounds had very similar effects on the kinetics and inhibition of lymphocyte growth. All compounds had complex effects on mitogenesis, causing enhanced, delayed, or inhibited tritiated thymidine incorporation. Although the total thiol contents of untreated T cells and B cells were found to be equivalent, the LPS response consistently was inhibited by lower concentrations than the Con A response, suggesting that B cells were more sensitive than T cells to thiol modification. To compare compounds the efficiency of inhibition was determined by functionally relating reductions in mitogenesis with reductions in thiol content of the cells. The compounds differed in inhibitory efficiency; thus, damage to some thiols must be more important than damage to others. CuP ablated mitogenesis with the least change in thiol content. Therefore, surface sulfhydryls appear critical in lymphocyte mito~ genesis. With all compounds inhibition of mitogenesis occurred over a very narrow range of thiol content, suggesting that the thiols important in inhibition were few in number relative to the total thiol content of the cell. *Address correspondence to Dr. D.A. Lawrence, Department of Microbiology and Immunology, A-68, Albany Medical College, Albany, N.Y. 12208. 0009-2797/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

138

Key words: Thiols -- Lymphocyte activation - Oxidative stress

INTRODUCTION In vitro the function of many proteins can be modified by alteration of their sulfhydryl moieties [1]. At the cellular level specific functions are also sensitive to thiol modulation, such as those of the cytoskeleton [2], the neutral amino acid transporter [3], Na ÷, K÷-ATPase [4], guanylate cyclase [5] and the ~-adrenergic receptor [6]. However, it is not clear which of the many sulfhydryl-dependent processes of the cell are the most sensitive to thiol modification, and therefore the most likely to be important in oxidatively stressed cells. The objective of this study was to determine the relative importance of different sulfhydryl constituents of the cell in lymphocyte mitogenesis. specific sulfhydryls of murine splenic lymphocytes were biochemically altered using sulfhydryl-modifying compounds. Cells were pretreated with four different compounds and washed prior to exposure to mitogens, so that the thiol status of the cells was altered before stimulation. The compounds differ in sulfhydryl reactivities and consequently different cell functions might be effected; thus the overall responsiveness of cells to mitogens, encompassing multiple passages through the cell cycle, was used to measure functional compromise in pretreated cells. Alterations in glutathione [7--10], surface thiols [11], and cellular thiols [12,13] modify lymphocyte functions. In this study, these different sulfhydryl constituents of the cell were compromised with BSO, CuP and NEM or H202, respectively. While all of these compounds are known inhibitors of lymphocyte mitogenesis, their relative effectiveness has not been determined; here, this determination was made by comparing the functional and structural damage each compound caused, by comparing reduced mitogenesis and altered thiol levels. MATERIALS AND METHODS Pre treatments Female CBA/J mice {Jackson Laboratory, Bar Harbor, ME} were used at 2--4 months o~ ~ge. Mice were maintained on laboratory chow and acidified, chlorinated water (pH 3.0) ad libitum. Splenic lymphocytes were enriched as previously described [14] and treated with CuP (1,10-phenanthroline, Sigma Chemical Co., St. Louis, MO; cupric sulfate, Fisher Scientific, Rochester, NY) at a 1 : 2 molar ratio of copper to phenanthroline [15], hydrogen peroxide (Sigma}, or NEM (Sigma) at a density of 1 x 108 cells/ml balanced salt solution at 37°C for 30 min. Cells were pretreated with BSO (Sigma) by incubation for 18 h at a density of 5 x 106 cells/ml of Minimum Essential Medium (Microbiological Associates) supplemented as described earlier [14] with 50

139 ~¢I 2-mercaptoethanol (2-ME)/5% fetal bovine serum, non-essential amino acids, sodium bicarbonate, gentamicin, and vitamins. Dose responses of cells protected by lipid radical scavengers were determined by pretreating cells for 15 min with optimal concentrations (5 ~M) of N-propyl gallate, butylated hydroxytoluene and butylated hydroxyanisole (NPG, BHT and BHA; Sigma) in 0.15% ethanol before adding the thiol-modifying compound, with the exception of BSO treatments, where scavengers were present for the 18-h pretreatment in 0.05% ethanol. Scavengers were not present after pretreatment, since inclusion in the 7-day culture had no additional effects on the responses. For all four compounds, cells were centrifuged after pretreatment and washed in BSS. One aliquot was taken for assay of total thiol content, and a second aliquot was cultured for assay of mitogenesis. Cell viability was assessed by mixing equal volumes of cells and 0.16% trypan blue (MCB Manufacturing Chemists, Norwood, OH), and enumerating cells that had taken up dye.

Mitogenesis assays After cells were pretreated and washed, they were cultured in supplemented MEM with 2 ~g/ml Con A (T cell mitogen) or 10 ~g/ml LPS (B cell mitogen) at a density of 1 x l0 s cells/ml (0.2 ml) in 96 well Costar clusters. Mitogenesis was measured as described previously [14], using tritiated thymidine deoxyribonucleoside ([SH]Thd, spec. act., 6.7 Ci/mmol; New England Nuclear, Boston, ME). The kinetics of incorporation after pretreatment were independently determined three times for each compound, in a total of 12 experiments. Thiol assays Total thiol levels and non-protein thiol levels were determined with a previously described fluorometric assay [16]. Total thiol levels were determined on aliquots of the same cells used in the mitogenesis assays, and non-protein thiol levels were determined in cells pretreated in separate experiments. Protein thiol levels were indirectly calculated by subtraction of non-protein thiol values from the total thiol values. Population studies Splenic lymphocytes were depleted of Thy 1" cells and/or slg* cells by incubation with antibody followed by complement [14]. Cells were either assayed for thiol content as described above, or phenotyped with flow cytometry. For phenotyping, cells were stained with 2 ~g antibody per 106 cells (monoclonal rat anti-mouse IgM, 331.22; monoclonal rat anti-Thy 1.2, 30-H12; or control rat IgG antibody) in PBS/2% FBS/0.1% azide for 30 min at 4°C, followed by a stain with fluoresceinated goat anti-rat Ig (Tago, Burlingame, CA). Cells were suspended in 5 ~g/ml propidium idodide in PBS, and analyzed on an EPICS V flow cytometer. Log integrated green fluorescence was analyzed from viable lymphocytes gated on log integrated red fluorescence, forward angle light scatter, and integrated 90 o light scatter.

140 RESULTS E a c h c o m p o u n d w a s t e s t e d for cell toxicity, and only c o n c e n t r a t i o n s of thiol m o d i f i e r s which c a u s e d m i n i m a l cell d e a t h w e r e used to a s s e s s dose effects on m i t o g e n e s i s . P r e t r e a t e d splenic l y m p h o c y t e s s h o w e d complex a l t e r a t i o n s in m i t o g e n e s i s e n c o m p a s s i n g enhanced, delayed, and r e d u c e d r e s p o n s i v e n e s s to b o t h Con A (Fig. 1) and L P S (Fig. 2), d e p e n d i n g on t h e c o n c e n t r a t i o n of s u l f h y d r y l modifier. All four c o m p o u n d s a f f e c t e d t h e kinetics and m a g n i t u d e of [SH]Thd incorporation. Generally, l o w e r c o n c e n t r a t i o n s e n h a n c e d r e s p o n s i v e n e s s , h i g h e r c o n c e n t r a t i o n s caused a delay, and y e t h i g h e r c o n c e n t r a t i o n s inhibited m i t o g e n e s i s . Selected doses a r e shown.

iO0

/ ~

A

B

80

~o 401

', I°'°IIIo ~II

o

80

•

.

.

.

.

.

.

.

.

~

/ A/

2o 0

, --....

I

I..'/\','. II ~.234567 ~23~56t DAY OF CULTURE

Fig. 1. Kinetics of [~H]Thd incorporation of pretreated, Con A-stimulated cells. Cells were pretreated with one of the four sulfhydryl modifiers, cultured with Con A, and pulsed for 6 h with 0.5 ~Ci [ZHrrhd on days 1--7 of culture. Each point is an average of three wells; the coefficient of variation was usually less than 10%. Incorporation of cells pretreated with selected inhibitory doses is shown, and the results are representative of three independent experiments. In ascending order ( ............. ), the concentrations were: (A) 0, 1.5, 2.0 and 2.5 ~Vl CuP; (B) 0, 1.0, 1.2 and 1.4 ~M NEM; (C) 0, 100, 125 and 150 mM BSO; and (D) 0, 30, 40 and 50 ~M H202.

141 70

so

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-"

: "Y-:'':

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60

=o

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D

50

20

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%

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_

DAY OF CULTURE Fig. 2. Kinetics of [SH]Thd incorporation of ])retreated, LPS-stimulated cells. Replicate samples of cells pretreated as in Fig. I were stimulated with LPS and pulsed with [sHrrhd on days I - - 7 of culture. Results are representative of three independent experiments. Symbols are as in Fig. I.

Because peak [~H]Thd incorporation was delayed with some concentrations of compounds, differences in incorporation on a single day were not representative of the entire response. To remove time as a variable, the sum of incorporation on all 7 days of culture was taken and expressed as a fraction of the summed incorporation of the untreated control (Fig. 3). Averages of three independent experiments are shown for each compound. Consistently the LPS response was inhibited at lower concentrations than the Con A response. The averaged Con A and LPS responses of Fig. 3 were compared using the T-test, but no significance was found because of the large interexperiment variation. Therefore a paired analysis was used to compare the two responses on an experiment-by-experiment basis. In all 12 experiments the LPS response was more sensitive than the Con A response. Graphical estimates of doses reducing total incorporation by 50% (IDa0) appear in Table I. The differences in IDa0 for the LPS and Con A responses

142 1.4" 1.2 1.0

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4

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0.2

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.

.

,

i .0

.

2.0

,

,

3.0

.

,

4.0

0:4

0.8

i.2

[NEM] (uM)

[CUP] [pM)

Z o

6

D n."~

"

1.2" 1.O;

0.8:

0.6: 0.40.2" 00

5"o

16o

[13S0] (mM)

T

150

o i'o 2o 3o 4"0 5"o so (H202) (pM)

Fig. 3. Dose response effects of CuP (A), NEM (B), BSO (C) and H~O~ (D) on mitogen-stimulated murine splenic lymphocytes. For cells p r e t r e a t e d with a given concentration of compound, the t~HJThd incorporation on days 1--7 was summed and expressed as a fraction of the summed incorporation of the untreated control. For Con A ( ) and LPS (- - -) the average of three independent experiments is shown, with the standard deviation.

were noted for each experiment; the frequency of these differences was significant at the P < 0.05 level using the non-parametric signs test [17]. The compounds also inhibited mitogenesis when cell numbers were determined as a measure of stimulation (not shownl. Cell viability was determined by trypan blue exclusion (Table II). With the exception of BSO, for most concentrations viability was greater than 900/0 immediately after pretreatment. After 24 h of culture viability declined further, particularly in the BSO and H202-treated cultures. The difference in sensitivity of the Con A and LPS responses might have been due to differing thiol levels in LPS and Con A responders. However, data in Table III indicate that this was not the case. T and B cell populations enriched to similar degrees had equivalent amounts of total thiols. Furthermore a population of Thy 1- sIg- cells also found in the splenic lymphocyte population showed total thiol levels equivalent to T cells and B cells.

143 TABLE I COMPARISON OF THE SENSITIVITY OF CON A AND LPS-STIMULATED CELLS The IDs concentrations of the Con A and LPS responses were determined graphically; three independent determinations were made for each compound. The sign of the difference of all paired observations was determined and all 12 were positive. The null hypothesis states t h a t there is no difference between the pairs, and t h a t the frequency of positive signs should be 0.5. Using the binomial distribution the frequency of all 12 being positive if the null hypothesis is true is (1/2)TM or 2.4 × 10-'. Therefore with P < 0.05 we can conclude t h a t the IDs0 for the Con A response is significantly greater than for the LPS response. Treatment

CuP

NEM

BSO

H202

Expt.

IDso

Difference

Con A

LPS

1 2 3

2.3 ~ 8.3 ~ 8.6 pM

1.6 ~ 2.7 ~ 2.5/~M

1 2 3

1.2 ~

+

0.7 ~

1.0 ~ 0.8 pM 0.6/~M

1.0 p M

+ +

+ +

+

1

125 mM

100 mM

+

2

150 m M

145 m M

+

3

140 mM

120 mM

+

1 2 3

44 I~M 41 ~ 40 ~

40 I~I 31 I~M 36 I~M

+ + +

TABLE II VIABILITY OF P R E T R E A T E D CELLS After p r e t r e a t m e n t cell viability was determined immediately or following 1 day of culture in supplemented medium without mitogens. Viability was determined using trypan blue exclusion. The average of t h r e e different experiments is shown, with the standard deviation. Treatment

Conc.

% Viable cells

Oh

24h

CuP

0/~M 4.0~

94.5 ± 91.0 ±

4 3

89.4 ± 85.1 ±

9 4

NEM

0~ 1.5/~M

94.0 ± 89.5 ±

3 5

88.4 ± 86.9 ±

8 3

BSO

0pM 150 ~

85.5 ± 4 52.2 ± 10

80.8 ± 50.6 ±

1 6

H202

0~ 701~I

94.5 ± 90.0 ±

90.0 ± 10 75.6 ± 1

4 1

144 TABLE III THIOL STATUS OF SPLENOCYTE POPULATIONS The total thiol content (TSH, with the standard deviation) and phenotype of splenoeyte subpopulations were determined after depleting splenic lymphoeytes of T cells and/or B cells with antiThyl and anti-surface Ig antibody, respectively, followed by complement. In neither experiment were there significant differences in thiol levels among the groups at the P = 0.05 level, using Scheffe's multiple range test. Depletion

Phenotype

TSH (amol/cell)

anti-Thyl

anti-sIg

%Thyl ÷

%sIg÷

O/oThyl-sIg-

Expt. 1

-+ -+

--+ +

45.3 0 83.6 0

29.7 75.0 2.2 4.4

25.0 25.0 14.2 95.6

1247 1170 1198 1271

± ± _+ ±

60 58 131 15

Expt. 2

-+ -+

--+ +

35.8 1.9 76.2 3.9

47.7 87.5 7.3 15.8

16.5 10.6 16.5 80.3

878 882 786 822

± ± ± ±

79 1 94 5

The total thiol assay was used to compare the damage caused by each of the four pretreatments. However, this assay was used to quantitate the total cellular thiols of a mixture of Con A and LPS responders, but [aH]Thd incorporation assays measured the individual responses. In order to compare the two assays, the fractional [3H]Thd incorporation of the Con A and LPS responses were averaged for each experiment, and the average of three experiments was taken for each compound. These appear in Fig. 4, with the total thiol levels. The average total thiol level of the twelve untreated controls was 2257 ± 644 amol/cell. As would be expected on the basis of their differing reactivities, the four compounds reduced total thiols to different extents. At concentrations which ablated incorporation of [3H~hd, total thiols were decreased 1 - 5 % by CuP, 200/0 by NEM, 35% by BSO and 40°/0 by H20 r The four compounds share the qualities of inhibiting mitogenesis and acting upon cellular thiol constituents. These qualities are directly compared in the functional relation shown in Fig. 5, where fractional thiol level and fractional incorporation (averaged for the LPS and Con A response) are plotted for each concentration of compound. CuP caused a precipitous decrease in mitogenesis without significant reductions in total thiol levels; with the other compounds, there were threshold levels of thiols, which when compromised resulted in a precipitous decrease in mitogenesis. These thresholds differed for the four compounds and were a reflection of the efficiency of thiol-based inhibition of mitogenesis. The order of efficiency of inhibition of mitogenesis was CuP, NEM, BSO, and H 2 0 2. Scheffe's multiple range test [17] was used to assess the significance of these differences. For a particular level of fractional incorporation, the corresponding cellular thiol levels were

145 .4

A

• 2, i .o, O.B: 0.6:

B

0.4 z o a: 0.2~ 0 U

1".0

Z 0

2".0 3".0 [CUP] (I.JM)

4".0

0

0:4

o~e

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[NEM] (~M) D

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.4 0

0

50

t00

[BSO] (mM}

t50

o i'o 2"o 3"o ~o 5o e'O 7"0 [H2~] (pH)

Fig. 4. Effect of the sulfhydryl modifiers on the mitogenic response and total thiol content. The Con A and LPS mitogenic dose responses were averaged within an experiment, and the average of three experiments is shown ( ). The total thiol content was determined in the same cells immediately after pretreatment. This also was expressed as a fraction of the total thiol content of the untreated control, and the average of three determinations is shown (- - -).

compared. Significant differences were found between CuP and BSO, and CuP and H202;there were no other significant differences. Because of the different chemical reactivities of the four compounds, different cellular constituents were probably compromised, which would cause the different thresholds seen. This possibility was explored by characterizing cellular damage in more detail. Non-protein thiols were determined in cells after pretreatment. The mean amount in the twelve controls was 458 ± 200 amol/cell, representing approximately 200/0 of the total thiols, in agreement with earlier results [16,18]. The pretreatments differentially compromised non-protein thiols (Fig. 6). CuP caused no decrease in non-protein thiols, consistent with its negligible effect on the total thiol content. At concentrations of thiol modifier which ablated [3H]Thd incorporation, non-protein thiols were reduced 750/0 by NEM, 20--30% by BSO, and 50°/0 by H202. When non-protein thiols were subtracted from the total thiols,

146 Z o

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CI:

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°

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!

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i

!

i

i

i

i

0.5 0.6 0.7 0.8 0.9 1.0 1.1 FRACTION CONTROL CELLULAR THIOLS

Fig. 5. Functional relation of incorporation and thiol content. For each concentration of the compound the averaged fractional incorporation is plotted with the corresponding averaged fractional thiol level. Error bars are identical to those in Fig. 4 but are omitted for clarity. Pictured are the relations for CuP ( o - - ) , NEM ( l - - --), BSO (O ) and HsOs ( A - - - - ) .

the compromises in protein thiols were CuP, 1 - 5 % ; NEM, 5 - 1 0 % ; BSO, 20%; and H202, 20%. Repair of thiol damage was assessed by assaying total thiol content of cells immediately after pretreatment, and after 6 h of culture in medium with or without mitogen. With each compound the dose response effects did not change after 6 h, indicating that free thiols were not regenerated by cellular repair mechanisms after pretreatment with any of the thiol modifiers (not shown). Because H202 c a n cause lipid peroxidation as well as sulfhydryl oxidation [19], lipid radical scavengers were used in all of the pretreatments to limit this damage. In the presence of BHT, BHA, and NPG, the H202 mitogenic dose response (incorporation vs. concentration) shifted to the right, indicating increased resistance to H202-mediated damage. The averages of four independent determinations of this rightward shift were 5.2 _+ 4.3/aM for the Con A response and 2.0 _+ 0.8 pM for the LPS response (Fig. 7); these shifts were both significant at the P = 0.05 level using the T-test. Damage of total thiols by H202 w a s unaffected by the lipid radical scavengers, since thiols were similarly decreased in the presence or absence of scavengers. Protection was not seen for [SH]Thd incorporation or total thiols with CuP, NEM, or BSO. The specificities of the CuP and BSO pretreatments were examined. We found that relatively high concentrations of BSO were required to inhibit mitogenesis in comparison to others [7,9]. Since cells were pretreated with

147

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00

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-~ ~

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(uM}

Fig. 6. Effect of sulfhydryl modifiers on non-protein thiol content. The non-protein thiol content of pretreated cells was determined and expressed relative to the untreated control. Averages of three experiments are shown with the standard deviation.

or r

1.4 i. 21

A

B

Z

Z

o!--4 0

o:

0.6 0.4 O. 21

°o

20

40

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Fig. 7. Effect of lipid radical scavengers on cells pretreated with H202and stimulated with (A) Con A, and (B) LPS. Cells were incubated for 15 rain with 5 ~ concentrations of NPG, BHT, and BHA in BSS before the indicated amounts of H20 ~ were added. Cells were incubated for 30 rain further, washed, and cultured as before. Pictured is the fractional incorporation of cells pretreated in the presence ( - - - - - ) or absence { ) of lipid radical scavengers, with the standard deviation.

148

BSO at high density for 18 h, a comparison was made between cells pretreated at either 1 X 106 or 5 x 106 cells/ml. There was little difference between the two in terms of total thiols, [SH]Thd incorporation, or viability at 0 and 24 h after pretreatment. Also use of the L-isomer rather than the D,L-isomer mixture shifted the dose responses only slightly to the left. Therefore, as discussed below, these concentrations were probably required because of low glutathione turnover in quiescent cells. The specificity of the CuP reactivity was also investigated, since phenanthroline might chelate essential metals or associate with cellular constituents [20]. To assess these possibilities cells were pretreated with phenanthroline in the absence of Cu2*; there was no inhibition of incorporation even at doses four times the inhibitory doses of CuP.

DISCUSSION Splenic lymphocytes pretreated with each of the four compounds showed enhanced, delayed or reduced responsiveness to T cell and B cell mitogens, depending on the concentration of sulfhydryl modulator. Others have reported augmentation by H202 Of the response of human peripheral blood lymphocytes to pokeweed mitogen [19] and murine splenic T lymphocytes to Con A [21], and also augmentation by NEM of the response of murine splenic T lymphocytes to Con A [22]. In light of the greater sensitivity of suppressor T cells to oxidative stress [22,23] enhancement may be due to preferential compromise of suppressor cell function in cells exposed to lower concentrations of sulfhydryl modulators [22]. Delayed incorporation was also seen at certain concentrations of each of the four sulfhydryl modifiers. This is in agreement with others who have found delayed proliferation kinetics with stimulated human peripheral blood lymphocytes cultured with H202[19] and NEM [12]. Others [8,10,13,24,25] have assessed the inhibitory effects of sulfhydryl-modulating compounds on one particular day of culture; if the kinetics of the response were affected as here, analyses of the sensitivity might be inaccurate. Mitogenesis was decreased not only when measured by [3H]Thd incorporation, but also by cell number and flow cytometric cell cycle analysis (manuscript in preparation), in agreement with others [9] who have examined the effects of BSO on lymphocyte mitogenesis. Therefore reduced incorporation was indicative of genuine inhibition of passage through S phase. Decreased mitogenesis is probably not due to inhibition of mitogen binding, since Con A binding is via saceharide residues and LPS binding is unsaturable [26]. Furthermore, Con A binding is unchanged in lymphocytes exposed to H202 [27], the glutathione depleters 2-cyclohexene-l-one [10] and diamide [13], and hyperbaric oxygen [25]. Thiol modulation of Con A-induced murine lymphocyte proliferation also does not seem to be dependent on the dose of Con A [23]. Splenic lymphocytes enriched by the methods used here are a mixture of many cell types. Our data indicate that T cells (Con A responders), B cells

149 (LPS responders), and non-T/non-B cells have similar amounts of total thiols. Non-protein thiols of T and B cells are also similar [18], yet the LPS response was consistently more sensitive to these treatments than the Con A response. This would suggest that available thiols are more critical for B cell than T cell responses. The greater sensitivity of the LPS response is consistent with other measures of the thiol status of T cells and B cells [22]: 2-ME enhances B cell proliferation more than T cell proliferation, B cells have more surface thiols, and B cells are more sensitive to sulfhydryl blockage and irradiation. In order to chemically dissect different thiol constituents of the cell, we utilized compounds with different chemical properties. However, panels of compounds with the same chemical reactivity but subtle differences in structure have been used to study the inhibitory effects of NEM [12], and the stimulatory effects of 2-ME [11]. In both cases the effect was dependent on the hydrophobicity of the compound. In this study, we sought to classify thiols into different categories (surface vs. total cellular, non-protein vs. total). With the four compounds the specific effects on the cells differed (summarized in Table IV). Different concentrations were required to inhibit mitogenesis, with NEM being the most effective on a molar basis. More relevant, though, is inhibition of mitogenesis expressed as a function of thiol inhibition. On this basis cell surface thiols were critical for responsiveness to mitogens, since CuP ablated mitogenesis while minimally effecting total thiols, non-protein thiols, or lipids. The other compounds caused more heterogeneous damage, since both non-protein and protein thiols were effected. In agreement with Hamilos and Wedner [7] we observed a slow depletion of glutathione, in that, after 18 h of incubation with BSO non-protein thiols had decreased only 20%. Treatment with BSO inhibits synthesis of glutathione; for the glutathione content of cells to decrease, preexisting glutathione must be utilized. In our studies BSO was used in a pretreatment before cells were

TABLE IV SUMMARY OF D A M A G E C A U S E D BY THIOL MODIFIERS Treatment

NPSH •

PSH b

Lipids ~

Via d

IDs"

T/B Ratio l

CuP NEM BSO

0 75 30 50

<5 10 20 20

-+

85.1 86.9 50.6 75.6

2.5 ~ 1.0/~M 135 mM 45 ~ I

1.37 1.19 1.18 1.18

H202

• Maximum % decrease in non-protein thiols relative to control. bMaximum % decrease in protein thiois relative to control. Indicated if protection was afforded by lipid radical scavengers. d Minimum % viable 24 h after p r e t r e a t m e n t . • ID~ of the averaged mitogenic r e s p o n s e s from Fig. 4. Ratio of the averaged ID60s of the Con A and LPS responses. A ratio g r e a t e r t h a n 1.0 indicates t h a t the Con A response is more r e s i s t a n t t h a n the L P S response.

150 exposed to mitogens. Thus the cells were quiescent, and probably utilization of glutathione was less than if the cells were activated. More extensive depletions have been achieved with lymphocytes by including BSO in culture during mitogenie stimulation [7,8]. Furthermore Fischman et al. [10] compared inhibitory effects of the glutathione depletor cyclohexene-l-one in a pretreatment vs. during mitogenic stimulation of human peripheral blood lymphocytes, and found inhibition only with depletion during mitogenesis. It should be noted that pretreatment with BSO was in the presence of 2-ME, but 2-ME does not substantially affect the glutathione-exhausting effect of BSO [8]. Only H202 caused lipid damage, as measured indirectly through prc~ tection studies with lipid radical scavengers. This damage was a minor component, since the protection afforded was small. The damage to thiols was more extensive. This is in contrast to the results of Freed et al. [19] who found that H202 treatments did not affect cellular thiols of human peripheral blood lymphocytes, and lipid radical scavengers afforded substantial protection. However in these studies H202 was added to the cell culture. Possibly iron in the serum used in the medium catalyzed the Fenton reaction, generating hydroxyl radical extracellularly. Membrane constituents would be very susceptible to damage under these conditions. In our studies, cells were pretreated with H202 in BSS in the absence of extraeellular iron. In this case hydroxyl radical was probably generated intracellularly from endogenous iron, and intracellular constituents such as thiols might have been more susceptible to hydroxyl radical attack. In spite of differing chemical reactivities and differing types of damage caused, the four sulfhydryl modifiers evoked similar functional responses in splenic lymphocytes. Dependent on dose, mitogenesis was enhanced, delayed or reduced; there was an indirect relation between damage to total cellular thiols and responsiveness to mitogens; and B cells were more sensitive than T cells. Preliminary evidence also indicates that at concentrations that ablate [SH]Thd incorporation, cells progress no further into the cell cycle than Go/Gla upon mitogenic stimulation. Possibly similar regulatory responses are being differentially provoked with each pretreatment. Responses such as synthesis of adenylylated nucleotides [28] or poly-ADP ribosylation [29] can be induced by oxidative stress and both responses have been proposed to modulate the cell cycle [30,31]. Alternatively, each pretreatment may affect the same cell process differentially. In light of preliminary evidence indicating little progression into the cell cycle after pretreatment and stimulation, we hypothesize that signal transduction, specifically phospholipid metabolism, protein kinase C activation, and/or calcium ion fluxes, are effected by all four treatments. Modulation of these thiol-related cellular processes by the four modifiers is currently under investigation. ACKNOWLEDGEMENT

This work was supported by NIH grant ES~03778.

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