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Evidence that lithium ions can modulate lectin stimulation of lymphoid cells by multiple mechanisms
CELLULAR
IMMUNOLOGY
58,372-384 (1981)
Evidence that Lithium Ions Can Modulate Lectin Stimulation Lymphoid Cells by Multiple Mechanisms
of
DAVID A. HART Department
of Microbiology.
University
Received April
of Texas Health Science Center. Dallas,
Texas 75235
28. 1980; accepted June 12. 1980
Inhibition of PHA stimulation of hamster lymph node cells by theophylline, DBcAMP, or indomethacin or PHA stimulation of thymocytes by theophylline or DBcAMP was partially reversed by addition of 10 mM LiCl to the cultures. Addition of LiCl to Con A-stimulated lymphoid cells treated with the same reagents did not alter the inhibition. In contrast, addition of 10 mM LiCl to Con A-stimulated cultures enhanced the inhibition induced by the Na,K ATPase inhibitor, ouabain. Like LiCI, this latter inhibitor was found to be effective in modulating stimulation only if added early in the culture. These data support the hypothesis that LiCl can modulate lymphocyte responsivenessat the level of cyclic nucleotide metabolism, as exemplified by PHA stimulation, or at the level of the Na,K ATPase, exemplified by Con A stimulation. The site of involvement of Li+ ion would appear to be dependent on the biochemical nature of the stimulating signal.
INTRODUCTION Lithium ion is widely utilized in psychiatry in the treatment of manicdepressive disease states (1). Although widely used, the biochemical mechanism of action of this ion is not well understood. It has been proposed to exert its effect via modulation of cyclic nucleotide metabolism by inhibition of adenylate cyclase (1, 2) or via modification of the activity of membrane Na,K ATPase resulting in altered Kt metabolism (1, 3). Recently several laboratories have reported that Li+ can modify the activity of lymphoid cells (4-10). Both She&man et a1.(4) and Gelfand et al. (5) provided evidence which supported the hypothesis that Li+ functioned by inhibiting adenylate cyclase. The former workers demonstrated that Li+ could reverse prostaglandin E, (PGe,) inhibition of phytohemagglutinin (PHA) stimulation of human peripheral blood lymphocytes and the latter group demonstrated that Li+ could reverse inhibition of lymphoid cell activity induced by drugs which elevate CAMP concentrations (theophylline, isoproteronal, indomethacin). In contrast to the above findings, evidence from this laboratory has indicated that the effect of Lif on Con A stimulation was linked to K’ metabolism, a result consistent with a role for Li’ in modulating the activity of the membrane Na,K ATPase (7). The present study was undertaken in an attempt to elucidate the mechanism of Li’ modulation of lectin stimulation of hamster lymphoid cells. Evidence is presented which indicates that Lif may function by modulating CAMP metabolism 372 000%8749/81/040372-13$02.00/O Copyright 0 1981 by Academic Pres, Inc. All rights of reproduction in any form rescrvcd.
LITHIUM
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during PHA stimulation of some lymphoid cell populations but not during Con A stimulation of the same populations. MATERIALS
AND METHODS
Materials
The lectins PHA-P and Con A were obtained from Difco Laboratories (Detroit, Mich.) and Sigma Chemical Company (St. Louis, MO.), respectively. Tissue culture medium RPM1 1640 was obtained from Associated Biomedic Systems (Buffalo, N.Y.). The absence of Li+ in the medium was confirmed by flame photometry. Lithium chloride was a reagent grade product of Mallinkrodt, Inc. (St. Louis, MO.). Theophylline was purchased from Calbiochem Corporation (San Diego, Calif.) while ouabain, dibutyryl cyclic AMP (DBcAMP), and dibutyryl cyclic GMP (DBcGMP) were purchased from Sigma. Indomethacin (Lot. L-590,226-OOA113) was a gift from Merck, Sharpe and Dohme Research Laboratories (Rahway, N.J.). Tritiated thymidine was obtained from Amersham-Searle Corporation (Arlington Heights, Ill.). All compounds, with the exception of indomethacin, were solubilized in tissue culture medium and sterilized by filtration (0.22 w Millipore filters). Indomethacin was solubilized in 95% ethanol and sterilized by the same procedure. Cell Culture
Female MHA hamsters (7-l 1 weeks of age) were obtained from Charles River Lakeview Hamster Colony (Vineland, N.J.). Lymphoid cell suspensions were prepared from isolated thymus and lymph node tissue as described previously (11). Cells were cultured for 72 hr in a humid atmosphere of air-lo% CO* at 37°C. Twenty-four hours prior to harvest 0.5-1.0 r*Ci C3H]thymidine ([3H]TdR, 5 Ci/ mM) was added to each culture. All assayswere performed in triplicate. Variation from the reported mean incorporation of [3H]TdR was less than 10%. The order of addition of reagents to cell cultures in the reported experiments was Li+ 15 min prior to compound to be tested (theophylline, DBcAMP, DBcGMP, indomethacin, ouabain) and then lectin 45 min later. Except where indicated, the concentrations of lectins employed (25-50 pg PHA and 1 pg Con A) were chosen because they were determined to be optimal concentrations in previous reports (6, 7, 11, 12). When indomethacin was tested, the final concentration of ethanol was approximately 1%. Sterile 95% ethanol was added to control cultures to the same concentration. Preliminary experiments indicated that 1% ethanol did not effect lectin stimulation of LNC. RESULTS Previous experience (6, 7) had demonstrated that the effects of LiCl on lymphocyte stimulation were maximal at 10 mM, therefore this concentration of Lit was employed in the present study. In addition, previous investigations (6, 7) revealed that the effects of Li+ on hamster lymphoid cells were unique to this ion; therefore the effects of other ions were not included in the present study except in specific instances.
374
DAVID A. HART
FIG. 1. Effect of theophylline, DBcAMP, and DBcGMP on lectin stimulation of lymph node cells. Cultures of LNC were stimulated with 50 pg PHA (A) or 1 /.rg Con A (B) for 72 hr. One hour prior to addition of the lectins, the indicated concentrations of theophylline, DBcAMP, or DBcGMP were added to triplicate cultures. The indicated values represent the mean incorporation of [jH/TdR into the triplicate cultures. Variation from the mean was less than 10%. The numbers above the bars indicate the percentage of control values.
Effect of Lithium on Theophylline, D&. Lectin Stimulation of LNC
and Indomethacin Inhibition
of
Theophylline is an inhibitor of CAMP phosphodiesterase. Inhibition of this enzyme leads to increased intracellular cyclic AMP levels due to inhibition of degradation. Addition of dibutyryl cyclic AMP to cells also leads to higher intracellular CAMP concentrations either by direct substitution for CAMP or by indirectly inhibiting CAMP phosphodiesterase (13, 14). Addition of these reagents, theophylline or DBcAMP, at concentrations of 10-5-10-3 M led to the differential inhibition of PHA stimulation of hamster LNC (Fig. 1). The response to PHA was inhibited 70-90s by 1O-3M theophylline while the Con A response was only inhibited by 2-10s by the same concentration of theophylline (Fig. 1). The PHA response was inhibited 50-80% by 10e4M DBcAMP and 90-95’S by 10m3M DBcAMP. In contrast, the Con A response was only inhibited 5-10 and 30-40s by the same concentrations of DBcAMP, respectively. The inhibition induced by the latter reagent, DBcAMP, was relatively specific since it was not induced by equivalent concentrations of dibutyryl cyclic GMP (Fig. 1). When cultures of LNC were supplemented with 10 mM LiCl, the inhibitory effects of DBcAMP and theophylline on PHA stimulation were reproducibly less dramatic (Fig. 2). Theophylline ( 10e3M) inhibited the PHA response 65-87’S in the control cultures and only 22-16s in the cultures with 10 mM LiCl. Lithium chloride was less effective in reversing DBcAMP-induced inhibition of PHA stimulation than in reversing theophylline-induced suppression (Fig. 2). At 10m4M DBcAMP, LiCl partially reversed the inhibition but at low3 iU DBcAMP, LiCl did not reverse the inhibition (Fig. 2). These results could be expected if 10e4M
LITHIUM
AND
LYMPHOCYTE
c
25pg PHA
4
WI PHA I PHA+iOmM
117
Control
ACTIVATION
-.
&
L-
Theophylline CM)
DBcAMP CM)
315
LICI
to” DBcAMP 04)
FIG. 2. Modulation of the inhibitory effects of theophylline and DBcAMP on PHA stimulation of LNC by LiCl. Cultures of LNC were stimulated with 25 pg PHA (upper) or 50 rg PHA (lower) in the presence of 10 mh4 LiCl (striped bars) or in the absence of LiCl (stippled bars). The order of addition of the reagents was as described under Materials and Methods. The indicated values represent the mean incorporation of [‘H]TdR into triplicate cultures at each point. Variation from the mean incorporation was less than 10%. The numbers above the bars indicate the percentage of control values.
DBcAMP exerted a primary effect on stimulation by inhibiting the CAMP phosphodiesterase and only weakly substituting for CAMP and if 10V3M DBcAMP inhibited stimulation by both inhibiting the phosphodiesterase and effectively substituting for cellular CAMP. In the presence of an effective exogenous source of CAMP, LiCl inhibition of adenylate cyclase would not have an observable effect on stimulation under these conditions. It should be noted that other experiments revealed that LiCl would also not relieve lo-’ M DBcAMP suppression of the Con A response (data not shown). Another compound which can indirectly modulate CAMP concentrations within cells is indomethacin. This compound inhibits prostaglandin synthesis (15) which in turn leads to decreased CAMP levels in the cell (16). Indomethacin is usually effective at micromolar concentrations although there is some tissue and species variation (15). Other enzyme systems such as the CAMP phosphodiesterase have been reported to be inhibited by higher concentrations of indomethacin (17). As She&man et al. (4) have reported that Li+ could reverse prostaglandin El-induced inhibition of PHA stimulation of human lymphocytes, it was of interest to determine the effect of prostaglandin-modulating drugs such as indomethacin on lectin stimulation of hamster lymphoid cells. Addition of 0.1-1.0 PM indomethacin (data not shown) or 10 FM indomethacin (Fig. 3) to LNC led to a 3-6s enhancement of the Con A response but not the PHA response. Enhancement was only observed at the low concentrations of indomethacin tested (l-10 FM). This slight enhancement with indomethacin was not drastically altered by addition of 10 mM LiCl to the cultures (Fig. 3). If indomethacin enhances stimulation by inhibiting prostaglandin synthesis, then the fact that there was only slight enhancement in the
376
DAVID A. HART
FIG. 3. Modulation of indomethacin inhibition of lectin stimulation of LNC by LiCl. Cultures of LNC were stimulated with 50 rg PHA (0, a) or 1 pg Con A (0, W) in the presence of 10 mM LiCl (open symbols) or in the absence of LiCl (closed symbols). The indicated concentrations of indomethacin were added 45 min prior to the lectin. The indicated values are expressed as the percentage of the [rH]TdR incorporated in the presence of indomethacin compared to that incorporated in the absence of indomethacin. The control cultures incorporated the following levels of [3H]TdR: PHA = 409,620 cpm, PHA + 10 mM LiCl = 401,390 cpm, Con A = 520,390 cpm, Con A + 10 mM LiCl = 464,930 cpm.
presence of this compound suggests that endogenous prostaglandins do not negatively regulate lectin stimulation of hamster lymphoid cells. While low concentrations of indomethacin did not exert a strongly positive effect on lectin stimulation, addition of higher concentrations of indomethacin (25-100 pLM) to cultures of LNC stimulated with either 50 pg PHA or 1 pg Con A led to a progressive inhibition of both responses (Fig. 3). However, the indomethacin inhibition profiles varied with each lectin. PHA stimulation was much more sensitive to indomethacin inhibition than was Con A stimulation. At 25 PM indomethacin, the PHA response was inhibited by approximately 50% while the Con A response was unaffected. Increasing the concentration of indomethacin to 50 PM led to a 40% inhibition of the Con A response while the PHA response was suppressed by greater than 90%. Concentrations of indomethacin greater than 75 PM were completely suppressive to both the Con A and PHA responses(Fig. 3). Lithium chloride (10 mM) was unable to reverse the inhibition profile for indomethacin suppression of the Con A response (Fig. 3). However, addition of 10 mM LiCl to the PHAstimulated cultures partially reversed the indomethacin inhibition. In the presence of Li+ the inhibition profile for this lectin was shifted such that it was nearly coincident with the Con A inhibition profile (Fig. 3). That is, in the presence of 10 mM LiCl, indomethacin inhibited the PHA and Con A responses to the same degree. From these results it would appear that the metabolic requirements for CAMP-dependent events during PHA stimulation differ from those required for Con A stimulation. Effect of Lithium on Theophylline and DBcAMP Inhibition of Lectin Stimulation of Thymocytes. From past experience it has been determined that certain parameters of stimulation of hamster lymphoid cells show tissue-specific characteristics (18). Therefore it was of interest to determine whether the findings obtained with LNC were comparable to those that could be obtained with thymocytes.
LITHIUM AND LYMPHOCYTEACTIVATION
311
6 PHP
105 16’ rb’ III Theophylline IM)
16 1o.d DBcAMP CM)
FIG. 4. Inhibition of PHA and Con A stimulation of thymocytes by theophylline and DBcAMP. Cultures of thymocytes were stimulated with 50 pg PHA (upper) or 1 pg Con A (lower) for 72 hr. One hour prior to addition of the lectin the indicated concentrations of theophylline or DBcAMPwere added to appropriate cultures. The indicated values represent the mean incorporation of [3H]TdR into triplicate assays at each point. Variation from the mean was less than 10%. The numbers above the bars indicate the percentage of controlvalues.
Hamster thymocytes were stimulated with an optimal concentration of PHA (50 rg/ml) or Con A (1 pg) and the effect of 1O-5-1O-3M theophylline or lo-‘10e3M DBcAMP was determined. Again PHA stimulation was differentially inhibited by these reagents (Fig. 4). However, in contrast to results obtained with LNC, Con A stimulation of thymocytes (Fig. 4) was reproducibly inhibited by concentrations of theophylline ( 10e3M) or DBcAMP ( 10-4-10-3 M) which were either noneffective or marginally effective in inhibiting Con A stimulation of LNC (Fig. 1). Addition of 10 mM LiCl to cultures of thymocytes led to a large enhancement of PHA stimulation (6) (Fig. 5). The observed enhancement of [3H]TdR incorporation was two- to threefold over that incorporated in the absenceof LiCl. Similar to results obtained with LNC (Fig. 2), it was also observed that LiCl could reduce the effectiveness of theophylline and DBcAMP to inhibit PHA stimulation (Table 1). When compared to stimulation in RPM1 1640 alone, neither 10m4M DBcAMP nor 10e4-5 X 10e4A4 theophylline were effective in inhibiting stimulation in the presence of 10 mM LiCl (Table 1). Compared to the same control, LiCl was ineffective in reversing inhibition induced by 10e3M DBcAMP and only marginally effective in reversing low3 M theophylline-induced inhibition of PHA stimulation. However, when the reference point is the [3H]TdR incorporated in RPM1 1640 + 10 mM LiCl, theophylline and DBcAMP were still very effective in inhibiting the response to PHA (Table 1). These results may indicate that LiCl enhancement of PHA stimulation of thymocytes involves more metabolic systems than those dependent on cyclic nucleotide metabolism. While 10 mM LiCl enhances PHA stimulation across the complete dose-response
318
DAVID A. HART 0 iOm3M Tbphylline
0.25
0
0.5 CDNCANAVALIN
IO
15
A (,q/mll
FIG. 5. Effect of LiCl on theophylline inhibition of Con A stimulation of thymocytes. Cultures of thymocytes were stimulated with the indicated concentrations of Con A in the absence of LiCl (0, 0) or in the presence of 10 mM LiCl (A, A). Theophylline, lo-’ M, was added to cultures (0, A) 45 min prior to the lectin. The indicated values represent the mean incorporation of [3H]TdR into triplicate assays at each point. Variation from the reported mean was less than 10%.
curve (6), addition of the same concentration of LiCl to cultures of thymocytes stimulated with Con A leads to enhancement of the response to low concentrations of Con A and inhibition of the response to higher concentrations of Con A (7) (Fig. 5). If this inhibitory effect on Con A stimulation was due to Lif inhibition TABLE 1 Effect of LiCl on Theophylline and DBcAMP Inhibition of PHA Stimulation of Thymocytes Percentage of control” DBcAMP (M)
Theophylline (M) Experiment 1
10 m&f LiCl
50 pg PHA 50 rg PHA
-
50 rg PHA 50 pegPHA
-
2
3 50 pg PHA 50 pg PHA
+
+
+
1O-3
1o-4
10-r
84 231 (70)
10 31 (9)
24 147 (45)
1
70 240 (76)
5 21 (7)
20
0.6
Control
lo-4
100 329 (100) 100 316 (100) 100 284 (100)
5 x 1o-4
17 (Z)
(i.6) 19 142 (50)
a Ratio of mean counts per minute [‘H]TdR incorporated into triplicate cultures/mean counts per minute [3H]TdR incorporated into control cultures X 100. The data are expressed either as percentage of the control cultures in RPM1 1640 with no LiCl or inhibitors or as percentage of the cultures in RPM1 1640 + 10 mMLiC1 with no inhibitors (numbers in parentheses). The counts per minute [3H]TdR incorporated into PHA (50 @g/ml)-stimulated cultures in RPM1 1640 with no inhibitors was: Expt 1 = 75,070 cpm, Expt 2 = 55,490 cpm, Expt 3 = 38,050 cpm.
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of adenylate cyclase, then one might expect the inhibition to be alleviated by agents which block intracellular CAMP degradation, such as theophylline, or reagents which may substitute for CAMP, such as DBcAMP. However, neither of these reagents could reverse LiCl-induced suppression of Con A stimulation. Theophylline, low3 M, was equally inhibitory to Con A stimulation across the complete dose-response curve (0.25-1.5 pg Con A/ml) in the absence (63 + 9% inhibition) or presence (64 ? 5% inhibition) of 10 mM LiCl (Fig. 5). Additional experiments also indicated that 10m4,5 X 10b4, or 10e3 M DBcAMP would not reverse LiClinduced suppression of stimulation by l-2 pg Con A nor would Li+ reverse inhibition of suboptimal (0.25 @g/ml) Con A stimulation induced by these concentrations of DBcAMP (data not shown). These results indicate that LiCl does not reverse theophylline or DBcAMP inhibition and that theophylline or DBcAMP do not reverse LiCl-induced suppression. The results therefore support the hypothesis that Li+ effects on Con A stimulation are due to modulation of biochemical events unrelated to cyclic nucleotide metabolism. Effect of Monovalent
Cations on Ouabain Inhibition
of Thymocyte
Stimulation
Ouabain is a glycoside which inhibits, in a competitive fashion, the membrane Na,K ATPase (reviewed in 19). Addition of this compound to cultures of lymphoid cells has been reported to inhibit early events of stimulation (20). Lithium ions have also been reported to modify the activity of this ATPase (3) as well as modulate early activation events during stimulation of hamster lymphoid cells by lectins (6, 7). Therefore it was of interest to determine if Li+ could exert an effect on ouabain modulation of lectin stimulation. Preliminary experiments indicated that lectin stimulation of hamster thymocytes was dramatically inhibited by concentrations of ouabain greater than 1Oe4M (Table 2). Both Con A and PHA stimulation was 90% inhibited by 2.5 X 10Y4M ouabain. This concentration of ouabain did not appear to be toxic for the cells as determined by trypan blue exclusion. Time of addition studies confirmed the conclusion that 2.5 x 10e4 M ouabain was not toxic for the cells as well as confirmed the results of Quastel and Kaplan (20) who found that ouabain was only effective if added early in the culture. As depicted in Fig. 6, it can be seen that 2.5 X 10e4M ouabain inhibited the Con A response by greater than 90% if added 1 hr prior to the Con A but it was less effective if added 5 hr after the Con A. If the time of addition was delayed until 24 hr after initiation of the cultures, 2.5 X lop4 M ouabain had little or no effect on Con A stimulation (Fig. 6). AS stated above, ouabain is a competitive inhibitor of the Na,K ATPase. The inhibitory effects of this glycoside can be reversed by increasing the K+ concentration (19). To determine whether this was also true for the observed effect of ouabain on Con A stimulation of hamster thymocytes, cells were cultured in RPM1 1640 (5 mM K+), RPM1 1640 + 25 mM KCl, or RPM1 1640 + 10 mM LiCl and then the inhibitory effects of either 10m4or 2 x 10e4M ouabain was investigated. AS shown in Fig. 7, addition of 25 mM KC1 to the medium was effective in reversing inhibition by 2 X 10m4M ouabain. Lithium chloride, on the other hand, enhanced the inhibitory effect of 2 X 10m4M ouabain. In fact 10 mM LiCl enhanced the marginal inhibition induced by 10e4M ouabain Fig. 7 (A). Experiments with PHA yielded similar results (data not shown). These data (Fig. 7) are analogous
380
DAVID
A. HART
TABLE Ouabain Inhibition
2
of Lectin Stimulation
of Thymocytes
Percentage of controP Lectin concentration (rglml)
Control”
0.1
1.0
2.0
2.5
Con A Expt 1 0.5 1.0
238,900 207,340
95 93
92 66
ND
15 12
Expt 2 0.5 1.0
209,490 160,830
ND
95 71
53 17
ND
PHA Expt 1 25 50 100
23,200 41,080 45.920
ND
88 77 76
ND
16 12 11
Expt 2 25 50 100
41,569 57,930 59,470
ND
63 65 55
21 21 18
Ouabain (M X 10m4)
a Mean incorporation of [‘H]TdR expressed as counts per minute. Variation from the mean was less than 10%. * Mean counts per minute [3H]TdR incorporated in the presence of ouabain/mean counts per minute [‘H]TdR incorporated in the absence of ouabain X 100.
to those of Osman et al. (21) who reported that intoxication of guinea pigs by digoxin or ouabain could be inhibited by K+ or Rb+ and potentiated by Lif. Therefore Li+ may modify some activity or function of a component of the Na,K ATPase which enhances the ability of ouabain to inhibit the enzyme. A second point from the data presented in Fig. 7 should also be emphasized. That is, the inhibitory effects of ouabain are some what dependent on the concentration of Con A utilized. Stimulation by 0.25 or 0.5 pg Con A was inhibited 2 to 5% by lop4 A4 ouabain while stimulation by 1 pg Con A was inhibited 28% by this concentration of ouabain. With 2 X 10e4 M ouabain, stimulation by 0.25 or 0.5 pg Con A was inhibited approximately 50% while stimulation by 1 pg Con A was inhibited 82%. This consistent result was not, however, observed in experiments with PHA (data not shown). Therefore it would appear that Con A may uniquely modify the Na,K ATPase, directly or indirectly, to enhance ouabain inhibition. If both Li+ and Con A modify the Na,K ATPase, this may be the basis for the observed Li+ inhibition of Con A stimulation at higher concentrations of the lectin (7) (Fig. 5). DISCUSSION Lithium ion has been proposed to exert its pharmacotherapeutic effects via alteration of cyclic nucleotide metabolism (1, 2) or through modification of the
LITHIUM
AND
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381
I
I Time of Addition (hn) . Control
-0
i.5
1.0
05 Con A Concentration
(pg/ml)
FIG. 6. Effect of time of addition on ouabain inhibition of Con A stimulation of thymocytes. Cultures of thymocytes were stimulated with the indicated concentrations of Con A in the absence of ouabain (0) or in the presence of 2.5 X 10F4 M ouabain added 1 hr prior to the Con A (0), 5 hr after the Con A (A) or 24 hr after the Con A (A). The indicated values represent the mean incorporation of [‘H]TdR into triplicate assays at each point. Variation from the mean was less than 10%.
activity of the Na,K ATPase (1, 3). Several published reports support both alternatives (l-6, 22-24). The data presented in this report also support both alternatives and lead one to 40
A.
Z6 30 ; 20
.-./I
E % 10
1
j_2. 0 6. 5 100
.Y'
B
-.
I
t
- .
.
‘\/------’ 0-
025
050
Con A C0ncentratv.x
1.00 (pg/ml)
FIG. 7. Modulation of ouabain inhibition of Con A stimulation of thymocytes by KC1 and LiCl. Thymocytes were cultured in RPM1 1640 (0). RPM1 1640 + 25 mM KC1 (m) or RPM1 1640 + 10 mM LiCl (A). Forty-five minutes prior to addition of the indicated concentrations of Con A, ouabain was added to appropriate cultures to a final concentration of 1 X 10m4M (A) or 2 X lo-“ M (B). The results are expressed as the percentage inhibition of [3H]TdR incorporation in the presence of ouabain compared to that incorporated in the absence of ouabain. Variation between samples at each point was less than 10%.
382
DAVID A. HART
conclude that there is no single biological effect that is mediated by Li+. The determination of the critical system which is modified is dependent on the nature of the stimulant. Stimulation of hamster LNC and thymocytes by PHA was very sensitive to inhibition by compounds which elevate CAMP concentrations, indicating that this lectin may stimulate lymphocytes by modulating CAMP-sensitive cellular metabolic events. PHA stimulation was inhibited by theophylline, DBcAMP, and indomethacin, all compounds which can lead to increases in intracellular CAMP concentrations. Addition of 10 mM LiCl to such inhibited cultures alleviated the inhibition of PHA stimulation. With the exception of DBcAMP, these results are similar to and consistent with the results of Shenkman et al. (4) and Gelfand et al. (5). These authors reported that Li+ could reverse inhibition of PHA stimulation of human peripheral blood lymphocytes by compounds which elevate CAMP concentrations. The exception between the data of Gelfand et al. (5) and that reported here is the ability of Li+ to reverse DBcAMP-induced inhibition. Gelfand et al. (5) reported that Li’ would not reverse DBcAMP inhibition, presumably since DBcAMP could get into the cells and function as CAMP. However, in the present report Li+ could reverse 10m4M DBcAMP-induced suppression of the PHA response, indicating that in hamster cells this compound may not inhibit the response by directly substituting for CAMP but may inhibit the response by an indirect mechanism such as by blocking intracellular CAMP degradation. It has been reported that the monobutyryl derivative of CAMP is an inhibitor of the phosphodiesterase and thereby could elevate CAMP levels indirectly (13, 14). In contrast to PHA, stimulation of hamster LNC by Con A was much more resistant to inhibition by agents which elevate intracellular CAMP levels. Addition of Li+ did not alter the response of Con A-stimulated cells to theophylline, DBcAMP, or indomethacin. From these results it would appear that stimulation of cells by this lectin may be through modification of CAMP-insensitive cellular events. It should be noted that stimulation of thymocytes by Con A was more sensitive to inhibition by the reagents tested but this inhibition was also resistant to reversal by Li+. The reason why there should be a difference between Con A and PHA stimulation is not known. The basis for the difference could reside in the biochemical mechanism of stimulation by the two lectins or could reside in the cell populations stimulated. There is some evidence that the two lectins stimulate different cell populations (25). The finding that Li+ does not appear to influence Con A stimulation at the cyclic nucleotide level leads to a search for alternative sites of action. The other postulated site of action of Li+ on cells is the Na,K ATPase (1, 3). This enzyme is responsible for maintenance of the potassium gradient across the plasma membrane (19). Interference with the maintenance of the gradient, by the use of ionophores (26, 27) or by blocking the Na,K ATPase (20) leads to inhibition of lymphocyte stimulation. Previously it was reported that Li+ effects on Con A stimulation were linked to K+ metabolism (7) although this ion will not substitute for K+ during stimulation (IO), The results obtained from the study of ouabain inhibition of Con A stimulation support the conclusion that Li+ effects are related to potassium metabolism i.e., the Na,K ATPase. Ouabain appeared to inhibit early events of stimulation which occur in the first 24 hr of culture (Fig. 6). This is the same time frame in which Li+ can modulate Con A (7) and PHA (6) stimulation. The finding that ouabain inhibition could be reversed by K+ and potentiated by Li+ is also
LITHIUM
AND
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ACTIVATION
383
indicative, but not conclusive evidence that Li+ can modulate the Na,K ATPase. Interestingly, mitogenic concentrations of Con A have been reported to increase Na,K ATPase activity of lymphoid cells, possibly by increasing the number of K+binding sites (discussed in 28). However, supra optimal concentrations of Con A have also been shown to inhibit the Na,K-dependent ATPase activity of isolated rat lymphoid cell membranes (29). The mechanism of this modulation is unknown; however one component of the isolated Na,K ATPase, distinct from the ouabainbinding component (30), is a glycoprotein which could possibly interact directly with Con A. Pertinent to this possibility is the finding of Resch, who has reported that lymphoid plasma membrane vesicles containing the Na,K ATPase preferentially bind to Con A-Sepharose (28). In conclusion, it would appear from the data that Li+ can exert an influence on lymphocyte stimulation by at least two mechanisms: at the level of cyclic nucleotide metabolism and/or at the level of the Na,K ATPase. The critical system modulated appears to be dependent on the nature of the stimulant. That is, Li+ apparently can act at the level of cyclic nucleotide metabolism and the Na,K ATPase, as evidenced by the results obtained with theophylline/indomethacin/DBcAMP and ouabain, respectively, on PHA stimulation or at the level of the Na,K ATPase, independent of cyclic nucleotide metabolism, as evidenced by the results obtained with the same reagents on Con A stimulation. If the latter conclusion is correct, then CAMP may not play a positive role during stimulation by Con A. As PHA and Con A, lectins with different carbohydrate specificities, both interact with a large number of membrane glycoproteins and glycolipids it is probable that they perturb a number or plasma membrane enzyme systems. Some of the signals are obviously mitogenic signals and others may or may not be necessary or essential for the completion of the proliferation sequence. From the results presented in this report, it would appear that Lif can modulate important metabolic systems but these systems may not be the primary mitogenic signals. However, the finding of differences between Li+ effects on PHA stimulation and Con A stimulation may provide a useful tool to dissect the molecular mechanism by which these lectins stimulate cells. ACKNOWLEDGMENTS The author wishes to thank Joan Stein-Streilein, and R. Jerrold Fulton for the critical review of the manuscript, Cheryl Hendrix for excellent technical assistance and Daisi Marcoulides for secretarial assistance in the preparation of the manuscript. This investigation was supported by Grants CA-24444 and AI-1 1851 from the National Institutes of Health, DHEW.
REFERENCES 1. Johnson, F. N., and Johnson, S. (Eds.), “Lithium in Medical Practice.” University Park Press, Baltimore, 1977. 2. Wang, Y.-C., Pandey, G. N., Mendels, J., and Frazer, A., Biochem. Pharmacol. 23, 845, 1974. 3. Robinson, J. D., Biochim. Biophys. Acta 413, 459, 1975. 4. Shenkman, L., Borkowsky, W., Holzman, R. S., and Shopsin, B., Clin. Immunol. Immunopathol. 10, 187, 1978. 5. Gelfand, E. W., Dosch, H.-M., Hasting, D., and Shore, A., Science 203, 365, 1979. 6. Hart, D. A., Exp. Cell Res. 119, 47, 1979. 7. Hart, D. A., Cell. Immunol. 43, 113, 1979. 8. Hart, D. A., Exp. Cell Res. 121, 419, 1979.
384
DAVID
A. HART
9. Hart, D. A., In “Molecular Basis of Immune Cell Function” (J. G. Kaplan, Ed.), p. 408. Elsevier/ North Holland, Amsterdam, 1979. 10. Hart, D. A., Cell. Immunol., in press. 11. Streilein, J. S., and Hart, D. A., Infect. Immunity 14, 463, 1976. 12. Hart, D. A., Stein-Streilein, J., and Eidels, L., Fed. Proc. 37, 2039, 1978. 13. Kaukel, E., and Hilz, H., Biochem. Biophys. Res. Commun. 46, 1011, 1971. 14. Hsie, A. W., Kawashima, K., O’Neill, J. P., and Schr&ler, C. H., J. Biol. Chem. 250, 984, 1975. 15. Ferreira, S. H., and Vane, J. R., In “The Prostaglandins” (P. W. Ramwell, Ed.), Vol. 2, p. 1. Plenum, New York, 1974. 16. Kahn, R. H., and Lands, W. E. (Eds.) “Prostaglandins and Cyclic AMP.” Academic Press, New York, 1973. 17. Flower, R. J., and Vane, J. R., Biochem. Pharmacol. 23, 1439, 1974. 18. Hart, D. A., Cell. Immunol. 32, 146, 1977. 19. Dahl, J. L., and Hokin, L. E., Ann. Rev. B&hem. 43, 327, 1974. 20. Quastel, M. R., and Kaplan, J. G., Nature (London) 219, 198, 1968. 21. Osman, F. H., Ammar, E. M., Afifi, A. M., and Ahmed, N. M., Japan. J. Exp. Med. 46, 1, 1975. 22. Dousa, T. P., Endocrinology 95, 1359, 1974. 23. Forn, J., and Valdecasas, F. G., B&hem. Pharmacol. 20, 2773, 1971. 24. Tobin, T., Akera, T., Han, C. S., and Brody, T. M., Mol. Pharmacol. 10, 501, 1974. 25. Stobo, J. D., Transplant. Rev. 11, 60, 1972. 26. Daniele, R. P., and Holian, S. K., Proc. Nat. Acad. Sci. USA 73, 3599, 1976. 27. Daniele, R. P., Holian, S. K., and Nowell, P. C. J. Exp. Med. 147, 571, 1978. 28. Resch, K. In “Molecular Basis of Immune Cell Function” (J. G. Kaplan, Ed.), p. 109, Elsevier/ North Holland, Amsterdam, 1979. 29. Novogrodsky, A., Biochim. Biophys. Acta 266, 343, 1972. 30. Forbush, B., Kaplan, J. H., and Hoffman, J. F., Biochemistry 17, 3667, 1978.
IMMUNOLOGY
58,372-384 (1981)
Evidence that Lithium Ions Can Modulate Lectin Stimulation Lymphoid Cells by Multiple Mechanisms
of
DAVID A. HART Department
of Microbiology.
University
Received April
of Texas Health Science Center. Dallas,
Texas 75235
28. 1980; accepted June 12. 1980
Inhibition of PHA stimulation of hamster lymph node cells by theophylline, DBcAMP, or indomethacin or PHA stimulation of thymocytes by theophylline or DBcAMP was partially reversed by addition of 10 mM LiCl to the cultures. Addition of LiCl to Con A-stimulated lymphoid cells treated with the same reagents did not alter the inhibition. In contrast, addition of 10 mM LiCl to Con A-stimulated cultures enhanced the inhibition induced by the Na,K ATPase inhibitor, ouabain. Like LiCI, this latter inhibitor was found to be effective in modulating stimulation only if added early in the culture. These data support the hypothesis that LiCl can modulate lymphocyte responsivenessat the level of cyclic nucleotide metabolism, as exemplified by PHA stimulation, or at the level of the Na,K ATPase, exemplified by Con A stimulation. The site of involvement of Li+ ion would appear to be dependent on the biochemical nature of the stimulating signal.
INTRODUCTION Lithium ion is widely utilized in psychiatry in the treatment of manicdepressive disease states (1). Although widely used, the biochemical mechanism of action of this ion is not well understood. It has been proposed to exert its effect via modulation of cyclic nucleotide metabolism by inhibition of adenylate cyclase (1, 2) or via modification of the activity of membrane Na,K ATPase resulting in altered Kt metabolism (1, 3). Recently several laboratories have reported that Li+ can modify the activity of lymphoid cells (4-10). Both She&man et a1.(4) and Gelfand et al. (5) provided evidence which supported the hypothesis that Li+ functioned by inhibiting adenylate cyclase. The former workers demonstrated that Li+ could reverse prostaglandin E, (PGe,) inhibition of phytohemagglutinin (PHA) stimulation of human peripheral blood lymphocytes and the latter group demonstrated that Li+ could reverse inhibition of lymphoid cell activity induced by drugs which elevate CAMP concentrations (theophylline, isoproteronal, indomethacin). In contrast to the above findings, evidence from this laboratory has indicated that the effect of Lif on Con A stimulation was linked to K’ metabolism, a result consistent with a role for Li’ in modulating the activity of the membrane Na,K ATPase (7). The present study was undertaken in an attempt to elucidate the mechanism of Li’ modulation of lectin stimulation of hamster lymphoid cells. Evidence is presented which indicates that Lif may function by modulating CAMP metabolism 372 000%8749/81/040372-13$02.00/O Copyright 0 1981 by Academic Pres, Inc. All rights of reproduction in any form rescrvcd.
LITHIUM
AND
LYMPHOCYTE
ACTIVATION
373
during PHA stimulation of some lymphoid cell populations but not during Con A stimulation of the same populations. MATERIALS
AND METHODS
Materials
The lectins PHA-P and Con A were obtained from Difco Laboratories (Detroit, Mich.) and Sigma Chemical Company (St. Louis, MO.), respectively. Tissue culture medium RPM1 1640 was obtained from Associated Biomedic Systems (Buffalo, N.Y.). The absence of Li+ in the medium was confirmed by flame photometry. Lithium chloride was a reagent grade product of Mallinkrodt, Inc. (St. Louis, MO.). Theophylline was purchased from Calbiochem Corporation (San Diego, Calif.) while ouabain, dibutyryl cyclic AMP (DBcAMP), and dibutyryl cyclic GMP (DBcGMP) were purchased from Sigma. Indomethacin (Lot. L-590,226-OOA113) was a gift from Merck, Sharpe and Dohme Research Laboratories (Rahway, N.J.). Tritiated thymidine was obtained from Amersham-Searle Corporation (Arlington Heights, Ill.). All compounds, with the exception of indomethacin, were solubilized in tissue culture medium and sterilized by filtration (0.22 w Millipore filters). Indomethacin was solubilized in 95% ethanol and sterilized by the same procedure. Cell Culture
Female MHA hamsters (7-l 1 weeks of age) were obtained from Charles River Lakeview Hamster Colony (Vineland, N.J.). Lymphoid cell suspensions were prepared from isolated thymus and lymph node tissue as described previously (11). Cells were cultured for 72 hr in a humid atmosphere of air-lo% CO* at 37°C. Twenty-four hours prior to harvest 0.5-1.0 r*Ci C3H]thymidine ([3H]TdR, 5 Ci/ mM) was added to each culture. All assayswere performed in triplicate. Variation from the reported mean incorporation of [3H]TdR was less than 10%. The order of addition of reagents to cell cultures in the reported experiments was Li+ 15 min prior to compound to be tested (theophylline, DBcAMP, DBcGMP, indomethacin, ouabain) and then lectin 45 min later. Except where indicated, the concentrations of lectins employed (25-50 pg PHA and 1 pg Con A) were chosen because they were determined to be optimal concentrations in previous reports (6, 7, 11, 12). When indomethacin was tested, the final concentration of ethanol was approximately 1%. Sterile 95% ethanol was added to control cultures to the same concentration. Preliminary experiments indicated that 1% ethanol did not effect lectin stimulation of LNC. RESULTS Previous experience (6, 7) had demonstrated that the effects of LiCl on lymphocyte stimulation were maximal at 10 mM, therefore this concentration of Lit was employed in the present study. In addition, previous investigations (6, 7) revealed that the effects of Li+ on hamster lymphoid cells were unique to this ion; therefore the effects of other ions were not included in the present study except in specific instances.
374
DAVID A. HART
FIG. 1. Effect of theophylline, DBcAMP, and DBcGMP on lectin stimulation of lymph node cells. Cultures of LNC were stimulated with 50 pg PHA (A) or 1 /.rg Con A (B) for 72 hr. One hour prior to addition of the lectins, the indicated concentrations of theophylline, DBcAMP, or DBcGMP were added to triplicate cultures. The indicated values represent the mean incorporation of [jH/TdR into the triplicate cultures. Variation from the mean was less than 10%. The numbers above the bars indicate the percentage of control values.
Effect of Lithium on Theophylline, D&. Lectin Stimulation of LNC
and Indomethacin Inhibition
of
Theophylline is an inhibitor of CAMP phosphodiesterase. Inhibition of this enzyme leads to increased intracellular cyclic AMP levels due to inhibition of degradation. Addition of dibutyryl cyclic AMP to cells also leads to higher intracellular CAMP concentrations either by direct substitution for CAMP or by indirectly inhibiting CAMP phosphodiesterase (13, 14). Addition of these reagents, theophylline or DBcAMP, at concentrations of 10-5-10-3 M led to the differential inhibition of PHA stimulation of hamster LNC (Fig. 1). The response to PHA was inhibited 70-90s by 1O-3M theophylline while the Con A response was only inhibited by 2-10s by the same concentration of theophylline (Fig. 1). The PHA response was inhibited 50-80% by 10e4M DBcAMP and 90-95’S by 10m3M DBcAMP. In contrast, the Con A response was only inhibited 5-10 and 30-40s by the same concentrations of DBcAMP, respectively. The inhibition induced by the latter reagent, DBcAMP, was relatively specific since it was not induced by equivalent concentrations of dibutyryl cyclic GMP (Fig. 1). When cultures of LNC were supplemented with 10 mM LiCl, the inhibitory effects of DBcAMP and theophylline on PHA stimulation were reproducibly less dramatic (Fig. 2). Theophylline ( 10e3M) inhibited the PHA response 65-87’S in the control cultures and only 22-16s in the cultures with 10 mM LiCl. Lithium chloride was less effective in reversing DBcAMP-induced inhibition of PHA stimulation than in reversing theophylline-induced suppression (Fig. 2). At 10m4M DBcAMP, LiCl partially reversed the inhibition but at low3 iU DBcAMP, LiCl did not reverse the inhibition (Fig. 2). These results could be expected if 10e4M
LITHIUM
AND
LYMPHOCYTE
c
25pg PHA
4
WI PHA I PHA+iOmM
117
Control
ACTIVATION
-.
&
L-
Theophylline CM)
DBcAMP CM)
315
LICI
to” DBcAMP 04)
FIG. 2. Modulation of the inhibitory effects of theophylline and DBcAMP on PHA stimulation of LNC by LiCl. Cultures of LNC were stimulated with 25 pg PHA (upper) or 50 rg PHA (lower) in the presence of 10 mh4 LiCl (striped bars) or in the absence of LiCl (stippled bars). The order of addition of the reagents was as described under Materials and Methods. The indicated values represent the mean incorporation of [‘H]TdR into triplicate cultures at each point. Variation from the mean incorporation was less than 10%. The numbers above the bars indicate the percentage of control values.
DBcAMP exerted a primary effect on stimulation by inhibiting the CAMP phosphodiesterase and only weakly substituting for CAMP and if 10V3M DBcAMP inhibited stimulation by both inhibiting the phosphodiesterase and effectively substituting for cellular CAMP. In the presence of an effective exogenous source of CAMP, LiCl inhibition of adenylate cyclase would not have an observable effect on stimulation under these conditions. It should be noted that other experiments revealed that LiCl would also not relieve lo-’ M DBcAMP suppression of the Con A response (data not shown). Another compound which can indirectly modulate CAMP concentrations within cells is indomethacin. This compound inhibits prostaglandin synthesis (15) which in turn leads to decreased CAMP levels in the cell (16). Indomethacin is usually effective at micromolar concentrations although there is some tissue and species variation (15). Other enzyme systems such as the CAMP phosphodiesterase have been reported to be inhibited by higher concentrations of indomethacin (17). As She&man et al. (4) have reported that Li+ could reverse prostaglandin El-induced inhibition of PHA stimulation of human lymphocytes, it was of interest to determine the effect of prostaglandin-modulating drugs such as indomethacin on lectin stimulation of hamster lymphoid cells. Addition of 0.1-1.0 PM indomethacin (data not shown) or 10 FM indomethacin (Fig. 3) to LNC led to a 3-6s enhancement of the Con A response but not the PHA response. Enhancement was only observed at the low concentrations of indomethacin tested (l-10 FM). This slight enhancement with indomethacin was not drastically altered by addition of 10 mM LiCl to the cultures (Fig. 3). If indomethacin enhances stimulation by inhibiting prostaglandin synthesis, then the fact that there was only slight enhancement in the
376
DAVID A. HART
FIG. 3. Modulation of indomethacin inhibition of lectin stimulation of LNC by LiCl. Cultures of LNC were stimulated with 50 rg PHA (0, a) or 1 pg Con A (0, W) in the presence of 10 mM LiCl (open symbols) or in the absence of LiCl (closed symbols). The indicated concentrations of indomethacin were added 45 min prior to the lectin. The indicated values are expressed as the percentage of the [rH]TdR incorporated in the presence of indomethacin compared to that incorporated in the absence of indomethacin. The control cultures incorporated the following levels of [3H]TdR: PHA = 409,620 cpm, PHA + 10 mM LiCl = 401,390 cpm, Con A = 520,390 cpm, Con A + 10 mM LiCl = 464,930 cpm.
presence of this compound suggests that endogenous prostaglandins do not negatively regulate lectin stimulation of hamster lymphoid cells. While low concentrations of indomethacin did not exert a strongly positive effect on lectin stimulation, addition of higher concentrations of indomethacin (25-100 pLM) to cultures of LNC stimulated with either 50 pg PHA or 1 pg Con A led to a progressive inhibition of both responses (Fig. 3). However, the indomethacin inhibition profiles varied with each lectin. PHA stimulation was much more sensitive to indomethacin inhibition than was Con A stimulation. At 25 PM indomethacin, the PHA response was inhibited by approximately 50% while the Con A response was unaffected. Increasing the concentration of indomethacin to 50 PM led to a 40% inhibition of the Con A response while the PHA response was suppressed by greater than 90%. Concentrations of indomethacin greater than 75 PM were completely suppressive to both the Con A and PHA responses(Fig. 3). Lithium chloride (10 mM) was unable to reverse the inhibition profile for indomethacin suppression of the Con A response (Fig. 3). However, addition of 10 mM LiCl to the PHAstimulated cultures partially reversed the indomethacin inhibition. In the presence of Li+ the inhibition profile for this lectin was shifted such that it was nearly coincident with the Con A inhibition profile (Fig. 3). That is, in the presence of 10 mM LiCl, indomethacin inhibited the PHA and Con A responses to the same degree. From these results it would appear that the metabolic requirements for CAMP-dependent events during PHA stimulation differ from those required for Con A stimulation. Effect of Lithium on Theophylline and DBcAMP Inhibition of Lectin Stimulation of Thymocytes. From past experience it has been determined that certain parameters of stimulation of hamster lymphoid cells show tissue-specific characteristics (18). Therefore it was of interest to determine whether the findings obtained with LNC were comparable to those that could be obtained with thymocytes.
LITHIUM AND LYMPHOCYTEACTIVATION
311
6 PHP
105 16’ rb’ III Theophylline IM)
16 1o.d DBcAMP CM)
FIG. 4. Inhibition of PHA and Con A stimulation of thymocytes by theophylline and DBcAMP. Cultures of thymocytes were stimulated with 50 pg PHA (upper) or 1 pg Con A (lower) for 72 hr. One hour prior to addition of the lectin the indicated concentrations of theophylline or DBcAMPwere added to appropriate cultures. The indicated values represent the mean incorporation of [3H]TdR into triplicate assays at each point. Variation from the mean was less than 10%. The numbers above the bars indicate the percentage of controlvalues.
Hamster thymocytes were stimulated with an optimal concentration of PHA (50 rg/ml) or Con A (1 pg) and the effect of 1O-5-1O-3M theophylline or lo-‘10e3M DBcAMP was determined. Again PHA stimulation was differentially inhibited by these reagents (Fig. 4). However, in contrast to results obtained with LNC, Con A stimulation of thymocytes (Fig. 4) was reproducibly inhibited by concentrations of theophylline ( 10e3M) or DBcAMP ( 10-4-10-3 M) which were either noneffective or marginally effective in inhibiting Con A stimulation of LNC (Fig. 1). Addition of 10 mM LiCl to cultures of thymocytes led to a large enhancement of PHA stimulation (6) (Fig. 5). The observed enhancement of [3H]TdR incorporation was two- to threefold over that incorporated in the absenceof LiCl. Similar to results obtained with LNC (Fig. 2), it was also observed that LiCl could reduce the effectiveness of theophylline and DBcAMP to inhibit PHA stimulation (Table 1). When compared to stimulation in RPM1 1640 alone, neither 10m4M DBcAMP nor 10e4-5 X 10e4A4 theophylline were effective in inhibiting stimulation in the presence of 10 mM LiCl (Table 1). Compared to the same control, LiCl was ineffective in reversing inhibition induced by 10e3M DBcAMP and only marginally effective in reversing low3 M theophylline-induced inhibition of PHA stimulation. However, when the reference point is the [3H]TdR incorporated in RPM1 1640 + 10 mM LiCl, theophylline and DBcAMP were still very effective in inhibiting the response to PHA (Table 1). These results may indicate that LiCl enhancement of PHA stimulation of thymocytes involves more metabolic systems than those dependent on cyclic nucleotide metabolism. While 10 mM LiCl enhances PHA stimulation across the complete dose-response
318
DAVID A. HART 0 iOm3M Tbphylline
0.25
0
0.5 CDNCANAVALIN
IO
15
A (,q/mll
FIG. 5. Effect of LiCl on theophylline inhibition of Con A stimulation of thymocytes. Cultures of thymocytes were stimulated with the indicated concentrations of Con A in the absence of LiCl (0, 0) or in the presence of 10 mM LiCl (A, A). Theophylline, lo-’ M, was added to cultures (0, A) 45 min prior to the lectin. The indicated values represent the mean incorporation of [3H]TdR into triplicate assays at each point. Variation from the reported mean was less than 10%.
curve (6), addition of the same concentration of LiCl to cultures of thymocytes stimulated with Con A leads to enhancement of the response to low concentrations of Con A and inhibition of the response to higher concentrations of Con A (7) (Fig. 5). If this inhibitory effect on Con A stimulation was due to Lif inhibition TABLE 1 Effect of LiCl on Theophylline and DBcAMP Inhibition of PHA Stimulation of Thymocytes Percentage of control” DBcAMP (M)
Theophylline (M) Experiment 1
10 m&f LiCl
50 pg PHA 50 rg PHA
-
50 rg PHA 50 pegPHA
-
2
3 50 pg PHA 50 pg PHA
+
+
+
1O-3
1o-4
10-r
84 231 (70)
10 31 (9)
24 147 (45)
1
70 240 (76)
5 21 (7)
20
0.6
Control
lo-4
100 329 (100) 100 316 (100) 100 284 (100)
5 x 1o-4
17 (Z)
(i.6) 19 142 (50)
a Ratio of mean counts per minute [‘H]TdR incorporated into triplicate cultures/mean counts per minute [3H]TdR incorporated into control cultures X 100. The data are expressed either as percentage of the control cultures in RPM1 1640 with no LiCl or inhibitors or as percentage of the cultures in RPM1 1640 + 10 mMLiC1 with no inhibitors (numbers in parentheses). The counts per minute [3H]TdR incorporated into PHA (50 @g/ml)-stimulated cultures in RPM1 1640 with no inhibitors was: Expt 1 = 75,070 cpm, Expt 2 = 55,490 cpm, Expt 3 = 38,050 cpm.
LITHIUM
AND
LYMPHOCYTE
ACTIVATION
379
of adenylate cyclase, then one might expect the inhibition to be alleviated by agents which block intracellular CAMP degradation, such as theophylline, or reagents which may substitute for CAMP, such as DBcAMP. However, neither of these reagents could reverse LiCl-induced suppression of Con A stimulation. Theophylline, low3 M, was equally inhibitory to Con A stimulation across the complete dose-response curve (0.25-1.5 pg Con A/ml) in the absence (63 + 9% inhibition) or presence (64 ? 5% inhibition) of 10 mM LiCl (Fig. 5). Additional experiments also indicated that 10m4,5 X 10b4, or 10e3 M DBcAMP would not reverse LiClinduced suppression of stimulation by l-2 pg Con A nor would Li+ reverse inhibition of suboptimal (0.25 @g/ml) Con A stimulation induced by these concentrations of DBcAMP (data not shown). These results indicate that LiCl does not reverse theophylline or DBcAMP inhibition and that theophylline or DBcAMP do not reverse LiCl-induced suppression. The results therefore support the hypothesis that Li+ effects on Con A stimulation are due to modulation of biochemical events unrelated to cyclic nucleotide metabolism. Effect of Monovalent
Cations on Ouabain Inhibition
of Thymocyte
Stimulation
Ouabain is a glycoside which inhibits, in a competitive fashion, the membrane Na,K ATPase (reviewed in 19). Addition of this compound to cultures of lymphoid cells has been reported to inhibit early events of stimulation (20). Lithium ions have also been reported to modify the activity of this ATPase (3) as well as modulate early activation events during stimulation of hamster lymphoid cells by lectins (6, 7). Therefore it was of interest to determine if Li+ could exert an effect on ouabain modulation of lectin stimulation. Preliminary experiments indicated that lectin stimulation of hamster thymocytes was dramatically inhibited by concentrations of ouabain greater than 1Oe4M (Table 2). Both Con A and PHA stimulation was 90% inhibited by 2.5 X 10Y4M ouabain. This concentration of ouabain did not appear to be toxic for the cells as determined by trypan blue exclusion. Time of addition studies confirmed the conclusion that 2.5 x 10e4 M ouabain was not toxic for the cells as well as confirmed the results of Quastel and Kaplan (20) who found that ouabain was only effective if added early in the culture. As depicted in Fig. 6, it can be seen that 2.5 X 10e4M ouabain inhibited the Con A response by greater than 90% if added 1 hr prior to the Con A but it was less effective if added 5 hr after the Con A. If the time of addition was delayed until 24 hr after initiation of the cultures, 2.5 X lop4 M ouabain had little or no effect on Con A stimulation (Fig. 6). AS stated above, ouabain is a competitive inhibitor of the Na,K ATPase. The inhibitory effects of this glycoside can be reversed by increasing the K+ concentration (19). To determine whether this was also true for the observed effect of ouabain on Con A stimulation of hamster thymocytes, cells were cultured in RPM1 1640 (5 mM K+), RPM1 1640 + 25 mM KCl, or RPM1 1640 + 10 mM LiCl and then the inhibitory effects of either 10m4or 2 x 10e4M ouabain was investigated. AS shown in Fig. 7, addition of 25 mM KC1 to the medium was effective in reversing inhibition by 2 X 10m4M ouabain. Lithium chloride, on the other hand, enhanced the inhibitory effect of 2 X 10m4M ouabain. In fact 10 mM LiCl enhanced the marginal inhibition induced by 10e4M ouabain Fig. 7 (A). Experiments with PHA yielded similar results (data not shown). These data (Fig. 7) are analogous
380
DAVID
A. HART
TABLE Ouabain Inhibition
2
of Lectin Stimulation
of Thymocytes
Percentage of controP Lectin concentration (rglml)
Control”
0.1
1.0
2.0
2.5
Con A Expt 1 0.5 1.0
238,900 207,340
95 93
92 66
ND
15 12
Expt 2 0.5 1.0
209,490 160,830
ND
95 71
53 17
ND
PHA Expt 1 25 50 100
23,200 41,080 45.920
ND
88 77 76
ND
16 12 11
Expt 2 25 50 100
41,569 57,930 59,470
ND
63 65 55
21 21 18
Ouabain (M X 10m4)
a Mean incorporation of [‘H]TdR expressed as counts per minute. Variation from the mean was less than 10%. * Mean counts per minute [3H]TdR incorporated in the presence of ouabain/mean counts per minute [‘H]TdR incorporated in the absence of ouabain X 100.
to those of Osman et al. (21) who reported that intoxication of guinea pigs by digoxin or ouabain could be inhibited by K+ or Rb+ and potentiated by Lif. Therefore Li+ may modify some activity or function of a component of the Na,K ATPase which enhances the ability of ouabain to inhibit the enzyme. A second point from the data presented in Fig. 7 should also be emphasized. That is, the inhibitory effects of ouabain are some what dependent on the concentration of Con A utilized. Stimulation by 0.25 or 0.5 pg Con A was inhibited 2 to 5% by lop4 A4 ouabain while stimulation by 1 pg Con A was inhibited 28% by this concentration of ouabain. With 2 X 10e4 M ouabain, stimulation by 0.25 or 0.5 pg Con A was inhibited approximately 50% while stimulation by 1 pg Con A was inhibited 82%. This consistent result was not, however, observed in experiments with PHA (data not shown). Therefore it would appear that Con A may uniquely modify the Na,K ATPase, directly or indirectly, to enhance ouabain inhibition. If both Li+ and Con A modify the Na,K ATPase, this may be the basis for the observed Li+ inhibition of Con A stimulation at higher concentrations of the lectin (7) (Fig. 5). DISCUSSION Lithium ion has been proposed to exert its pharmacotherapeutic effects via alteration of cyclic nucleotide metabolism (1, 2) or through modification of the
LITHIUM
AND
LYMPHOCYTE
ACTIVATION
381
I
I Time of Addition (hn) . Control
-0
i.5
1.0
05 Con A Concentration
(pg/ml)
FIG. 6. Effect of time of addition on ouabain inhibition of Con A stimulation of thymocytes. Cultures of thymocytes were stimulated with the indicated concentrations of Con A in the absence of ouabain (0) or in the presence of 2.5 X 10F4 M ouabain added 1 hr prior to the Con A (0), 5 hr after the Con A (A) or 24 hr after the Con A (A). The indicated values represent the mean incorporation of [‘H]TdR into triplicate assays at each point. Variation from the mean was less than 10%.
activity of the Na,K ATPase (1, 3). Several published reports support both alternatives (l-6, 22-24). The data presented in this report also support both alternatives and lead one to 40
A.
Z6 30 ; 20
.-./I
E % 10
1
j_2. 0 6. 5 100
.Y'
B
-.
I
t
- .
.
‘\/------’ 0-
025
050
Con A C0ncentratv.x
1.00 (pg/ml)
FIG. 7. Modulation of ouabain inhibition of Con A stimulation of thymocytes by KC1 and LiCl. Thymocytes were cultured in RPM1 1640 (0). RPM1 1640 + 25 mM KC1 (m) or RPM1 1640 + 10 mM LiCl (A). Forty-five minutes prior to addition of the indicated concentrations of Con A, ouabain was added to appropriate cultures to a final concentration of 1 X 10m4M (A) or 2 X lo-“ M (B). The results are expressed as the percentage inhibition of [3H]TdR incorporation in the presence of ouabain compared to that incorporated in the absence of ouabain. Variation between samples at each point was less than 10%.
382
DAVID A. HART
conclude that there is no single biological effect that is mediated by Li+. The determination of the critical system which is modified is dependent on the nature of the stimulant. Stimulation of hamster LNC and thymocytes by PHA was very sensitive to inhibition by compounds which elevate CAMP concentrations, indicating that this lectin may stimulate lymphocytes by modulating CAMP-sensitive cellular metabolic events. PHA stimulation was inhibited by theophylline, DBcAMP, and indomethacin, all compounds which can lead to increases in intracellular CAMP concentrations. Addition of 10 mM LiCl to such inhibited cultures alleviated the inhibition of PHA stimulation. With the exception of DBcAMP, these results are similar to and consistent with the results of Shenkman et al. (4) and Gelfand et al. (5). These authors reported that Li+ could reverse inhibition of PHA stimulation of human peripheral blood lymphocytes by compounds which elevate CAMP concentrations. The exception between the data of Gelfand et al. (5) and that reported here is the ability of Li+ to reverse DBcAMP-induced inhibition. Gelfand et al. (5) reported that Li’ would not reverse DBcAMP inhibition, presumably since DBcAMP could get into the cells and function as CAMP. However, in the present report Li+ could reverse 10m4M DBcAMP-induced suppression of the PHA response, indicating that in hamster cells this compound may not inhibit the response by directly substituting for CAMP but may inhibit the response by an indirect mechanism such as by blocking intracellular CAMP degradation. It has been reported that the monobutyryl derivative of CAMP is an inhibitor of the phosphodiesterase and thereby could elevate CAMP levels indirectly (13, 14). In contrast to PHA, stimulation of hamster LNC by Con A was much more resistant to inhibition by agents which elevate intracellular CAMP levels. Addition of Li+ did not alter the response of Con A-stimulated cells to theophylline, DBcAMP, or indomethacin. From these results it would appear that stimulation of cells by this lectin may be through modification of CAMP-insensitive cellular events. It should be noted that stimulation of thymocytes by Con A was more sensitive to inhibition by the reagents tested but this inhibition was also resistant to reversal by Li+. The reason why there should be a difference between Con A and PHA stimulation is not known. The basis for the difference could reside in the biochemical mechanism of stimulation by the two lectins or could reside in the cell populations stimulated. There is some evidence that the two lectins stimulate different cell populations (25). The finding that Li+ does not appear to influence Con A stimulation at the cyclic nucleotide level leads to a search for alternative sites of action. The other postulated site of action of Li+ on cells is the Na,K ATPase (1, 3). This enzyme is responsible for maintenance of the potassium gradient across the plasma membrane (19). Interference with the maintenance of the gradient, by the use of ionophores (26, 27) or by blocking the Na,K ATPase (20) leads to inhibition of lymphocyte stimulation. Previously it was reported that Li+ effects on Con A stimulation were linked to K+ metabolism (7) although this ion will not substitute for K+ during stimulation (IO), The results obtained from the study of ouabain inhibition of Con A stimulation support the conclusion that Li+ effects are related to potassium metabolism i.e., the Na,K ATPase. Ouabain appeared to inhibit early events of stimulation which occur in the first 24 hr of culture (Fig. 6). This is the same time frame in which Li+ can modulate Con A (7) and PHA (6) stimulation. The finding that ouabain inhibition could be reversed by K+ and potentiated by Li+ is also
LITHIUM
AND
LYMPHOCYTE
ACTIVATION
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indicative, but not conclusive evidence that Li+ can modulate the Na,K ATPase. Interestingly, mitogenic concentrations of Con A have been reported to increase Na,K ATPase activity of lymphoid cells, possibly by increasing the number of K+binding sites (discussed in 28). However, supra optimal concentrations of Con A have also been shown to inhibit the Na,K-dependent ATPase activity of isolated rat lymphoid cell membranes (29). The mechanism of this modulation is unknown; however one component of the isolated Na,K ATPase, distinct from the ouabainbinding component (30), is a glycoprotein which could possibly interact directly with Con A. Pertinent to this possibility is the finding of Resch, who has reported that lymphoid plasma membrane vesicles containing the Na,K ATPase preferentially bind to Con A-Sepharose (28). In conclusion, it would appear from the data that Li+ can exert an influence on lymphocyte stimulation by at least two mechanisms: at the level of cyclic nucleotide metabolism and/or at the level of the Na,K ATPase. The critical system modulated appears to be dependent on the nature of the stimulant. That is, Li+ apparently can act at the level of cyclic nucleotide metabolism and the Na,K ATPase, as evidenced by the results obtained with theophylline/indomethacin/DBcAMP and ouabain, respectively, on PHA stimulation or at the level of the Na,K ATPase, independent of cyclic nucleotide metabolism, as evidenced by the results obtained with the same reagents on Con A stimulation. If the latter conclusion is correct, then CAMP may not play a positive role during stimulation by Con A. As PHA and Con A, lectins with different carbohydrate specificities, both interact with a large number of membrane glycoproteins and glycolipids it is probable that they perturb a number or plasma membrane enzyme systems. Some of the signals are obviously mitogenic signals and others may or may not be necessary or essential for the completion of the proliferation sequence. From the results presented in this report, it would appear that Lif can modulate important metabolic systems but these systems may not be the primary mitogenic signals. However, the finding of differences between Li+ effects on PHA stimulation and Con A stimulation may provide a useful tool to dissect the molecular mechanism by which these lectins stimulate cells. ACKNOWLEDGMENTS The author wishes to thank Joan Stein-Streilein, and R. Jerrold Fulton for the critical review of the manuscript, Cheryl Hendrix for excellent technical assistance and Daisi Marcoulides for secretarial assistance in the preparation of the manuscript. This investigation was supported by Grants CA-24444 and AI-1 1851 from the National Institutes of Health, DHEW.
REFERENCES 1. Johnson, F. N., and Johnson, S. (Eds.), “Lithium in Medical Practice.” University Park Press, Baltimore, 1977. 2. Wang, Y.-C., Pandey, G. N., Mendels, J., and Frazer, A., Biochem. Pharmacol. 23, 845, 1974. 3. Robinson, J. D., Biochim. Biophys. Acta 413, 459, 1975. 4. Shenkman, L., Borkowsky, W., Holzman, R. S., and Shopsin, B., Clin. Immunol. Immunopathol. 10, 187, 1978. 5. Gelfand, E. W., Dosch, H.-M., Hasting, D., and Shore, A., Science 203, 365, 1979. 6. Hart, D. A., Exp. Cell Res. 119, 47, 1979. 7. Hart, D. A., Cell. Immunol. 43, 113, 1979. 8. Hart, D. A., Exp. Cell Res. 121, 419, 1979.
384
DAVID
A. HART
9. Hart, D. A., In “Molecular Basis of Immune Cell Function” (J. G. Kaplan, Ed.), p. 408. Elsevier/ North Holland, Amsterdam, 1979. 10. Hart, D. A., Cell. Immunol., in press. 11. Streilein, J. S., and Hart, D. A., Infect. Immunity 14, 463, 1976. 12. Hart, D. A., Stein-Streilein, J., and Eidels, L., Fed. Proc. 37, 2039, 1978. 13. Kaukel, E., and Hilz, H., Biochem. Biophys. Res. Commun. 46, 1011, 1971. 14. Hsie, A. W., Kawashima, K., O’Neill, J. P., and Schr&ler, C. H., J. Biol. Chem. 250, 984, 1975. 15. Ferreira, S. H., and Vane, J. R., In “The Prostaglandins” (P. W. Ramwell, Ed.), Vol. 2, p. 1. Plenum, New York, 1974. 16. Kahn, R. H., and Lands, W. E. (Eds.) “Prostaglandins and Cyclic AMP.” Academic Press, New York, 1973. 17. Flower, R. J., and Vane, J. R., Biochem. Pharmacol. 23, 1439, 1974. 18. Hart, D. A., Cell. Immunol. 32, 146, 1977. 19. Dahl, J. L., and Hokin, L. E., Ann. Rev. B&hem. 43, 327, 1974. 20. Quastel, M. R., and Kaplan, J. G., Nature (London) 219, 198, 1968. 21. Osman, F. H., Ammar, E. M., Afifi, A. M., and Ahmed, N. M., Japan. J. Exp. Med. 46, 1, 1975. 22. Dousa, T. P., Endocrinology 95, 1359, 1974. 23. Forn, J., and Valdecasas, F. G., B&hem. Pharmacol. 20, 2773, 1971. 24. Tobin, T., Akera, T., Han, C. S., and Brody, T. M., Mol. Pharmacol. 10, 501, 1974. 25. Stobo, J. D., Transplant. Rev. 11, 60, 1972. 26. Daniele, R. P., and Holian, S. K., Proc. Nat. Acad. Sci. USA 73, 3599, 1976. 27. Daniele, R. P., Holian, S. K., and Nowell, P. C. J. Exp. Med. 147, 571, 1978. 28. Resch, K. In “Molecular Basis of Immune Cell Function” (J. G. Kaplan, Ed.), p. 109, Elsevier/ North Holland, Amsterdam, 1979. 29. Novogrodsky, A., Biochim. Biophys. Acta 266, 343, 1972. 30. Forbush, B., Kaplan, J. H., and Hoffman, J. F., Biochemistry 17, 3667, 1978.