CELLULAR
IMMUNOLOGY
Modulation
43, 113- 122 (1979)
of Concanavalin A Stimulation of Hamster Ceils by Lithium Chloride1 DAVID
Department
of Microbiology,
University
Lymphoid
A. HART
of Texas Health Science Center at Dallas, Dallas, Texas 75235
Received
September 6. 1978
Addition of LiCl (l-25 mM) to serum-free cultures of MHA hamster thymocytes, lymph node cells, or splenocytes stimulated with concanavalin A had a biphasic effect on [3H]thymidine incorporation. These concentrations of LiCl enhanced stimulation of [3H]thymidine incorporation by suboptimal levels of concanavalin A but inhibited stimulation of optimal and supraoptimal concentrations of concanavalin A. This effect was specific for Li+ since it was not observed when similar concentrations of Na+, K+, or Mg*+ were added to cultures stimulated by concanavalin A. The inhibitory effect of LiCl on concanavalin A stimulation was not reversed by addition of Na+, Ca2+, Mg2+, or Ca*+ + Mg2+ to the cultures. Significant reversal of LiCl inhibition of stimulation was observed when KC1 was added to the cultures. However none of the ions tested blocked the Li-induced enhancement of [3H]thymidine incorporation in the presence of suboptimal concentrations of concanavalin A.
INTRODUCTION It has been proposed that early during mitogen stimulation of lymphocytes are related to changes in the intracellular concentration of various ions. The ions that have been implicated in such events include Ca2+ (1, 2) and K+ (3-5). Recently it was reported that Mn2+, a putative Ca2+antagonist, inhibited an early event during stimulation of lymphocytes by mitogens (6,7). In addition zinc ions alone have been shown to be mitogenic for both human (8, 9), hamster (lo), and guinea pig (10) lymphoid cells. Another ion which has been shown to have biologic activity in a variety of systems is Li+. In fact this ion is widely utilized pharmacologically in the field of psychiatry for the treatment of manic depressive states (1 1- 13). In addition to an effect on behavior, a variety of side effects of Li+ have been noted in both man and experimental animals (12, 13). These include obesity, muscle weakness, and alterations in the immune system and lymphoid cells. These later findings include involution of the thymus of both normal and adrenylectomized mice (14), concentration of in vivo administered Li+ in the cortex of the thymus (15), lymphopenia (16, 17), and the appearance of antinuclear antibodies in a significant percentage of treated patients compared to age-matched controls (18). It is also interesting to note that in the latter study, Li+ treatment of a patient with an ’ This investigation was supported by National Institutes of Health Lymphocyte Biology AI-11851.
Grant
113 0008-8749/79/030113-10$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproductionin any form reserved.
114
DAVID
A. HART
autoimmune disease, systemic lupus erythematous (SLE), led to an exacerbation of the disease (18). The pharmacotherapeutic mechanisms of Li+ are still unclear. This ion has been reported to modulate the activity of a large number of enzymes (19) as well as have an effect on the distribution of other ions such as Mg2+, Ca2+, and Na+ (19). A considerable literature has accumulated which has led to the idea that the important systems that are modulated by Li+ are related to either cyclic nucleotide metabolism (20, 21) or membrane ATPases (22, 23) possibly by interfering with Mg2+ (21) or K+-dependent events (23). These latter postulated sites of action of Li+, coupled with the above-described effects of LiCl on lymphoid cells, raised the possibility that Li+ could exert a direct effect on lymphocyte activation or function. In the present report evidence is presented that LiCl can modulate serum-free stimulation of hamster lymphoid cells by the T-cell mitogen, concanavalin A. MATERIALS
AND METHODS
Materials. Concanavalin A (Con A) was obtained from Sigma Chemical Company. The salts NaCl, KCl, CaCl,, MgC12,and LiCl were all reagent grade and were obtained from Mallinkrodt Inc. Stock solutions of salts, 1 M, were prepared in glass distilled water and sterilized by filtration through sterile 0.22~pm filters (Millipore Corp.). Tissue culture medium RPMI-1640 containing penicillin and streptomycin was obtained from Associated Biomedic Systems. Tritiated thymidine ([3H]TdR), 2-5 Ci/mM, was obtained from Amersham-Searle Corporation. Cell cultures. Lymphoid tissues (spleen, lymph nodes, and thymuses) were removed from 7- to lZweek-old female MHA hamsters (Charles River Lakeview Hamster Colony) and single-cell suspension prepared. Viability was determined by the trypan blue exclusion test. The cells, 5 x lo6 viable cells in 1 ml RPMI-1640, were cultured under serum-free conditions for 72 hr in a humid atmosphere of air-lo% CO, as described previously (24). Twenty-four hours prior to harvest, 0.5-1.0 $Ji[3H]TdR was added to each culture. The cultures were harvested on glass filters and incorporated [3H]TdR was determined as previously described (24). All assays were performed in triplicate and the variation from the mean incorporation was less than 10% in all reported experiments. Addition of ions. Unless otherwise indicated in individual experiments, appropriate concentrations of salts were added 1 hr prior to the mitogen. In multiple ion experiments, the LiCl was added 2-3 min after the first salt(s) was added to the cultures. RESULTS Effect of LiCl on Con-A Stimulation of Thymocytes Addition of LiCl to unstimulated cultures of thymocytes did not alter [3H]TdR incorporation over the concentration range of I-25 mM LiCl. However addition of LiCl to cultures of thymocytes stimulated with 0.1-2.0 pg Con A led to a dose dependent inhibition of this mitogenic response (Fig. 1). The inhibition observed was dependent on both the concentration of LiCl and the concentration of Con A added to the cultures. That is, at low suboptimal concentrations of Con A, addition
Li AND LYMPHOCYTE
115
STIMULATION
CcnA CONCENTRATION
(pg/ml)
FIG. 1. Modulation by LiCl or Con A stimulation of thymocytes. MHA thymocytes were stimulated with the indicated concentrations of Con A in RPMI-1640 alone (0) or in the presence of 1 mM LiCl (0), 5 m&f LiCl (A), 10 n&f LiCl (A), or 25 mkf LiCl (e). The indicated values represent the mean incorporation of [3H]TdR into triplicate assays. The variation from the mean incorporation was less then 10%.
of LiCl to the cultures led to an enhanced incorporation of [3H]TdR while the same concentrations of LiCl inhibited optimal stimulation by Con A (Fig. 1). Experiments utilizing Con A concentrations of 0.001-0.25 pg, indicated that addition of LiCl(5- 10 m&f) to cultures treated with submitogenic doses of Con A did not lead to stimulation of [3H]TdR incorporation (Fig. 2). Thus LiCl enhanced suboptimal stimulation by Con A but did not permit stimulation by submitogenic stimuli. The finding that stimulation by suboptimal concentrations of Con A was enhanced by LiCl, as well as trypan blue exclusion test data, indicated that the concentrations of LiCl tested were not toxic for the cells.
-0
o.ml
001
O.C6
Con A CONCENTRATION
025
03 (pghl
1
FIG. 2. The effect of LiCl on suboptimal Con A stimulation of MHA thymocytes. Cells were stimulated with the indicated concentrations of Con A in RPMI-1640 alone (0) or in the presence of 5 mM LiCl (0), or 10mM LiCl (A). The indicated values represent the net mean incorporation of 13H]TdR into triplicate assays. The variation from the mean incorporation was less than 10%.
116
DAVID A. HART
+24 TIME
OF ADDlTlON
OF LiCl
(hrsl
FIG. 3. The effect of time of addition on LiCl inhibition of Con A stimulation of MHA thymocytes. Cells were stimulated with 1 pg Con A (0,O) or 2 pg Con A (&A) in RPMI-1640 (control). At the indicated times 10 mM LiCl (.,A) or 25 mM LiCl (0,A) was added to appropriate cultures. The time of addition of the Con A was considered T = 0. The indicated values represent the mean incorporation of [3H]TdR into triplicate assays. The variation from the mean incorporation was less than 10%.
This latter conclusion was also supported by time of addition experiments. Lithium chloride was added to cultures of thymocytes at various times after stimulation with 1 and 2 pug Con A. As the data depicted in Fig. 3 indicate, concentrations of LiCl which were strongly inhibitory when added at the initiation of the culture, rapidly lost their ability to inhibit the response to Con A. The most dramatic loss in inhibition occurred within the first 24 hr of culture. When LiCl was added after 48 hr of culture, little or no inhibition of the Con A response was observed (Fig. 3). This latter finding eliminated the trivial explanation that the inhibition observed was due to LiCl interference with [3H]TdR transport and incorporation into the cells. Effect of LiCl on Con A Stimulation
of Lymph Node Cells and Splenocytes
Similar to results obtained with thymocytes, addition of LiCl to cultures of lymph node cells (Fig. 4) or splenocytes (data not shown), stimulated with suboptimal concentrations of Con A, led to an enhancement of [3H]TdR incorporation. However, in contrast to results obtained with thymocytes, stimulation of lymph node cells by optimal concentrations of Con A was not strongly inhibited by 10 mM LiCl (Fig. 4). When the concentration of LiCl was raised to 25 rnM, extensive inhibition of the Con A response was observed. These results indicated that stimulation of lymph node cells may be more refractory to the effects of LiCl than thymocyte populations. Also in contrast to the findings obtained with thymocytes, addition of 5- 10 mM LiCl to unstimulated cultures of lymph node cells, and to a lessor extent splenocytes, led to a two- to six-fold stimulation of [3H]TdR incorporation. Specificity
of the Effect of LiCl on Lymphocyte
Stimulation
To determine whether the observed effects of LiCl on Con A stimulation of lymphoid cells, particularly thymocytes, was specific for this ion, several other ions
Li AND LYMPHOCYTE
ConA
STIMULATION
CONCENTRATION
117
(pg/ml)
FIG. 4. Modulation by LiCl of Con A stimulation of MHA lymph node cells. Cells were cultured in the presence of the indicated concentrations of Con A in RPMI-1640 alone (@) or after addition of 10 mM LiCl (O), or 25 rnkf LiCl (A). The indicated values represent the mean incorporation of [3H]TdR into triplicate assays. The variation from the mean incorporation was less than 10%.
were also tested. Previously it has been shown that 50 @4 ZnCl, (10) or 50- 100pM MnCl, (6) inhibits Con A responses from all tissues tested over the entire dose range of Con A. Ions tested in the present study which had no inhibitory effect on Con A stimulation of thymocytes were NaCl(25 mM), KC1 (25 mkf), MgC& (25 m&f), and CaCl, (1.6 mM). From these results it was concluded that the inhibitory effect of LiCl on Con A stimulation of thymocytes was relatively specific for this ion and was not due to an osmotic effect. While the ions tested did not inhibit Con A stimulation of lymphoid cells, some of those tested, particularly Mg2+, did enhance stimulation by suboptimal concentrations of Con A (discussed in more detail below). Reversal of the Inhibitory Effect of LiCl by Other Ions It is believed that Li+ exerts some of its biological effects through antagonism with other ions in enzyme systems. This ion has been postulated to interfere with the activity of adenyl cyclase via an antagonism with Mg2+(21) or to modify the activity of NaK-ATPases through antagonism with K+ (23). To determine whether Li+ was exerting an inhibitory effect on Con A stimulation of thymocytes by displacing one or more of these other ions from a system essential for activation of thymocytes, attempts were made to reverse the inhibitory effect of Li+ on Con A stimulation with Mg2+, Ca2+, Mg2+ + Ca2+, Na+, K+, and K+ + Mg2+. In contrast to LiCI, MgC12(5-25 n&f) did not increase the [3H]TdR incorporated into unstimulated cultures of splenocytes or lymph node cells. In addition, the inhibition of Con A stimulation by LiCl was not drastically altered by the presence or absence of 25 rn44 MgCl, (Fig. 5) although there was some reversal of the inhibition induced by 10 mk! LiCl noted in the presence of 25 mkf MgC12.However, when compared to the control (Fig. 5a), addition of 25 mM MgCi, (Fig. 5b) to cultures of thymocytes stimulated with Con A led to an enhancement of [3H]TdR incorporation over the entire dose range of Con A; but in particular at the 0.1 wg Con A level where a 15-fold enhancement of [3H]TdR incorporation was observed.
118
DAVID A. HART
ConA UJNCENlRATlON
(pghnl)
FIG. 5. The effect of MgCl, on LiCl modulation of Con A stimulation. Thymocytes were cultured in RPMI-1640(A) or in RPMI-1640 + 25 mb4 MgC& (B). The cells were then stimulated with the indicated concentrations of Con A with no further additions (0) or after addition of either 10 mM LiCl(0) or 25 mM LiCl (A). The indicated values represent the mean incorporation of [3H]TdR into triplicate assays. The variation from the mean incorporation was less than 10%.
Similar results were obtained from cultures of lymph node cells or splenocytes (data not shown). It should be noted that the enhancement of suboptimal stimulation of r3H]TdR incorporation observed when MgC& or LiCl was added to cultures was further enhanced when 10 mk! LiCl + 25 rnkf MgC& was added but there was no induction of stimulation by submitogenic doses of Con A (Fig. 6). At the 0.1 ,ug Con A level, the [3H]TdR incorporated in the presence of Mg + Li was 253-fold enhanced over that incorporated with no additional Mg or Li. Therefore rather than an antagonism in this system there appears to be an additive effect of the two ions. Results similar to those described above for 25 mM MgC& (Fig. 5) were obtained when attempts were made to reverse LiCl inhibition with either 1.6 m&f Ca2+or 25 mA4 MgC12 + 1.6 mM CaCl, (data not shown). That is suboptimal Con A stimulation was enhanced in the presence of these ions but LiCl-induced inhibition was nor reversed. From these three sets of experiments it would appear that LP was not exerting an inhibitory/enhancement effect on Con A stimulation via antagonism with either Mg2+ or Ca2+. Lithium has also been proposed to be an antagonist of K+ ions (23). This latter ion has been postulated to be important in lymphocyte stimulation by several investigators (3-5). Rapid alterations in the concentration of this ion have been reported to occur after mitogen stimulation (3-5). This is believed to occur via
Li AND LYMPHOCYTE
STIMULATION
ConA CONCENTRATION
119
(pg/ml)
FIG. 6. Potential of suboptimal Con A stimulation by LiCl and M&l,. Thymocytes were stimulated with the indicated suboptimal concentrations of ConA in RPMI-1640 alone (0) or after addition of 10mM LiCl(O), 25 nGt4MgCI, (A), or 10n&f LiCl + 25 mM MgCl, (A). The indicated values represent the net mean incorporation of [3H]TdR into triplicate assays. The variation from the mean was less than 10%.
modification of the activity of a membrane NaK-ATPase (3). Inhibition of this ATPase with the specific inhibitor ouabain blocks early stimulation events (3, 25, 26). Therefore experiments were preformed to evaluate the effect of other monovalent cations on thymocyte stimulation. Preliminary experiments indicated that addition of 25 mM NaCl to cultures of thymocytes stimulated with Con A (0.1-2.0 pg) had no effect on either stimulation or LiCl-induced inhibition (data not shown). Additional attempts were then made to reverse LiCl inhibition of Con A stimulation with KCl. As seen in Fig. 7, addition of IO-25 mM KC1 to Con A stimulated cultures had no effect on stimulation but addition of these concentrations of KC1 to LiCl-inhibited cultures led to a significant reversal of the observed inhibition. The reversal of inhibition was most notable at the l-pg Con A level. At this level of Con A there was a dose-dependent reversal by 10and 25 mM KC1 of the inhibition induced by 25 n&f LiCl (Fig. 7). In the control cultures, stimulation by l-pg Con A was inhibited 70% by 25 mM LiCl while in cultures with 10 and 25 mM KC1 this concentration of LiCl inhibited the l-Fg Con A response by only 30% and 8%, respectively. As Li+ has been postulated to exert biological effects via antagonism with Mg2+ or K+, attempts were then made to reverse LiCl inhibition of Con A stimulation with a combination of K+ + Mg2+. When such experiments were performed with 12.5 mM MgCl, + 12.5 mM KC1 or 20 mM MgC& + 20 mM KCl, there was no dramatic reversal of LiCl inhibition (data not shown). The inhibition observed in the presence of both Mg2+ and K+ was not significantly different from that seen in the presence of K+ alone (Fig. 7). This finding indicated that Li+ was not exerting an effect via antagonism with two separate essential systems, one Mg2+ dependent and the other K+ dependent.
120
DAVID A. HART
120 / / 60 ,I’.,’ 60 x,p ::40 ./?/ 0
I. .r-.‘\, ,’,Ii ,.I ‘.’
0.1 1.0 2.0 CatA CONCENTRATloN (#nil
FIG. 7. The effect of KC1 on LiCl modulation of Con A stimulation. Thymocytes were cultured in RPMI-1640, RPMI-1640 + 10 mM KCI, or RPMI-1640 + 25 nGt4KCl. The cells were then stimulated with the indicated concentrations of Con A with no further additions (0) or after addition of 10mM LiCl (0), or 25 m44 LiCl(A). The indicated values represent the mean incorporationof [3H]TdRinto triplicate assays. The variation from the mean was less than 10%.
DISCUSSION The results presented in this study demonstrate that LiCl can have a direct effect on lymphocyte stimulation. The points that may be concluded from the data include the following: (i) LiCl potentiates suboptimal Con A stimulation; (ii) LiCl inhibits optimal and supraoptimal Con A stimulation; (iii) LiCl acts to modify early stimulation events; (iv) the action of LiCl is unique to lithium; and (v) thymocytes exhibit a sensitivity to the effects of LiCl not seen with lymph node cells or splenocytes. The mechanism by which Li+ exerts a biological effect in most systems is not well understood. However, the possibilities that have been raised include alteration of cyclic nucleotide metabolism (20,21) or modification of membrane enzymes such as membrane NaK-ATPases (22, 23). Both of these possibilities are relevant to lymphocyte stimulation since both cyclic nucleotides [reviewed in (27, 28)] and changes in specific ion flux (l-5) have been implicated as early activation events.
Li AND LYMPHOCYTE
STIMULATION
121
The results obtained in the present study would tend to support the latter hypothesis rather than the former cyclic nucleotide hypothesis. This conclusion is based on the finding that LiCl-induced inhibition of stimulation was partially reversed by K+ (Fig. 7) but not Mg2+ (Fig. 5) and the fact that Li+ + Mg2+ yielded additive enhancement of suboptimal stimulation (Fig. 6). As Li+ has been postulated to alter cyclase activity via antagonism with Mg2+ (21), it is doubtful that Li+ modulates lymphocyte activation via this mechanism since Li+ and Mg2+ appeared to act in a positive fashion in the present report. The hypothesis that Li+ acts by altering specific ion flux is very attractive for a variety of reasons. It has been proposed, and supported by experimental observations, that interaction of lectins such as Con A with lymphoid cells leads to rapid changes in ion flux across the plasma membrane. The two ions which have been implicated are K+ (3-5) and Ca2+(1,2). Modification of the flux of the former ion, K+, is thought to be mediated via membrane-bound NaK-dependent ATPases (4) and the importance of this enzyme activity has been demonstrated with the specific inhibitor, ouabain (3, 25, 26). Addition of this inhibitor to cultures of lymphocytes stimulated with lectins leads to inhibition of early activation events (25). In addition, Li+ has been shown to activate some NaK-dependent ATPases, even in the presence of saturating concentrations of K+ (23). Apparently Li+ displaces K+ from some sites on the enzyme which regulate the activity of the enzyme. In the present study K+ ions were the only effective cations which modified the inhibitory phase of the Li+ effect on Con A stimulation. If indeed specific ion flux is necessary for mitogenic stimulation then suboptimal stimulation could be limited by the ability of the stimulant to induce a primary mitogenic event and then modify rate limiting systems. This statement is based on the inability of Li+ to induce stimulation by itself or to induce mitogenesis by submitogenic concentrations of Con A, and assuming the enzyme is active in the membrane of the unstimulated cell, changes in the activity of the enzyme alone would not be a primary mitogenic signal. However, the enhancement by ions (LiCl + MgCl,) of stimulation by suboptimal concentrations of Con A, to levels comparable to that obtained with optimal concentrations of Con A, indicates that possibly the additional Con A is not inducing a primary mitogenic signal but rather is modifying rate-limiting steps in the activation process. The enhancing effect of Lit on suboptimal Con A stimulation could then result from activation of such a rate-limiting event. Therefore addition of LiCl to optimally stimulated cultures led to inhibition because of “over-activation” of the appropriate activities. A second issue raised by the data presented is related to the apparent unique sensitivity to LiCl of thymocyte stimulation. This data provides evidence that the previously reported involution of the thymus following in vivo administration of LiCl could be due to a direct effect of Li+ on thymic tissue (14). This conclusion is also supported by the work of Nelson ef al. (15) who reported that the thymic cortex concentrates in vivo-administered Li+. These observations raise the possibility, first proposed by Perez-Cruet and Dancey (14), that chronic administration of LiCl could lead to altered immune responses. The accumulated data, presented in this report and previously discussed under Introduction, strongly suggest that one ofthe immune systems which could be modified by LiCl is the suppressor T-cell system. Reports from several laboratories have indicated that suppressor T cells are short lived [reviewed in (29)], and a decline in suppressor-cell function has been
122
DAVID A. HART
postulated to accompany age and thymic involution (30-32). Such a hypothesis would predict that the progressive lymphopenia (16, 17) observed during chronic Li+ administration is due to a decline in a T-cell population. Further analysis of these hypotheses for the effect of Li+ on immune functions is currently in progress. ACKNOWLEDGMENTS The author wishes to thank Janet Frazier for excellent technical assistance during the course of this investigation, Daisi Marcoulides for secretarial assistance in the preparation of the manuscript, and Drs. Joan Stein-Streilein and R. Jerrold Fulton for their review of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
Whitney, R., and Sutherland, R. M., Cell Zmmunol. 5, 137, 1972. Freedman, M. H., Raff, M. C., and Gomperts, B., Nature 255, 378, 1975. Quastel, M. S, Dow, D. S., and Kaplan, J. G. Proc. Leucocyre Cult. Conf. 5, 97, 1970. Averdunk, R., and Lauf, P. K., Exp, Cell Res. 93, 331, 1975. Segel, G. B., and Lichtman, M. A., J. Clin. Invest. 58, 1358, 1976. Hart, D. A., Exp. Cell Res. 113, 139, 1978. Hart, D. A., Fed. Proc. 37, 1550, 1978. Kirchner, H., and Rilhl, H., Exp, Cell Res. 61, 229, 1970. Berger, N. A., and Skinner, A. M., J. Cell Biol. 61, 45, 1974. Hart, D. A., Zfect. Zmmunol. 19, 457, 1978. Kupfer, D. J., (Ed.), “Lithium and Psychiatry Journal Articles.” Medical Examination Publ. Co., Flushing, N.Y., 1971. Johnson, F. M., and Johnson, S., (Eds.), “Lithium in Medical Practice.” University Park Press, Baltimore, 1977. Jefferson, J. W., and Greist, J. H. (Eds.), “Primer of Lithium Therapy.” Williams & Wilkins, Baltimore, 1977. Perez-Cruet, J., and Dancey, J. T., Experientiu 33, 646, 1977. Nelson, S. C., Herman, M. M., Bensch, K. G., Sher, R., and Barchas, J. D., Exp. Mol. Pathol. 25, 38, 1976. Perez-Cruet, J., Dancey, J. T., and Waite, J., In “Lithium and Medical Practice.” (F. N. Johnson and S. Johnson Eds.), p. 271, University Park Press, Baltimore, 1977. Radomski, J., Fuyat, H. N., Nelson, A. A., and Smith, P. K., J. Pharmacol. 100, 429, 1950. Presley, A. P., Kahn, A., and Williamson, N., bit. Med. J. 2(6030), 280, 1976. Birch, N. J., In “Lithium in Medical Practice.” (F. N. Johnson and S. Johnson, Eds.), p.89, University Park Press, Baltimore, 1977. Geisler, A., Klysner, R., and Thams, P. In “Lithium and Medical Practice.” (F. N. Johnson and S. Johnson, Eds.), p. 159, University Park Press, Baltimore, 1977. Wang, Y-C., Pandey, G. N., Mendels, J., and Frazer, A., Biochem Pharm. 23, 845, 1974. Glen, A. I. M. In “Lithium in Medical Practice.” (F. N. Johnson and S. Johnson, Eds.), p. 183, University Park Press, Baltimore, 1977. Robinson, J. D., Biochim, Biophys, Acta 413, 459, 1975. Streilein, J. S., and Hart, D. A., Znfec. Zmmunol. 14, 463, 1976. Quastel, M. R., and Kaplan, J. G. Nature (London) 219, 198, 1968. Negendank, W. G., and Collier, C. R., Exp. Cell Res. 101, 31, 1976. Wedner, H. J., and Parker, C. W., Prog. Allergy 20, 195, 1976. Oppenheim, J. J., and Rosenstreich, D. L., Prog. Allergy 20, 65, 1976. Moller, G. (Ed.), Transplant. Rev. 26, 1975. Okumura, K., and Tada, T., J. Zmmunol. 106, 1019, 1971. Kerbel, R. S., and Eidinger, D., Eur. J. Zmmunol. 2, 114, 1972. Allision, A. C., Denman, A. M., and Barnes, R. D., Lancer 1, 135, 1971.
IMMUNOLOGY
Modulation
43, 113- 122 (1979)
of Concanavalin A Stimulation of Hamster Ceils by Lithium Chloride1 DAVID
Department
of Microbiology,
University
Lymphoid
A. HART
of Texas Health Science Center at Dallas, Dallas, Texas 75235
Received
September 6. 1978
Addition of LiCl (l-25 mM) to serum-free cultures of MHA hamster thymocytes, lymph node cells, or splenocytes stimulated with concanavalin A had a biphasic effect on [3H]thymidine incorporation. These concentrations of LiCl enhanced stimulation of [3H]thymidine incorporation by suboptimal levels of concanavalin A but inhibited stimulation of optimal and supraoptimal concentrations of concanavalin A. This effect was specific for Li+ since it was not observed when similar concentrations of Na+, K+, or Mg*+ were added to cultures stimulated by concanavalin A. The inhibitory effect of LiCl on concanavalin A stimulation was not reversed by addition of Na+, Ca2+, Mg2+, or Ca*+ + Mg2+ to the cultures. Significant reversal of LiCl inhibition of stimulation was observed when KC1 was added to the cultures. However none of the ions tested blocked the Li-induced enhancement of [3H]thymidine incorporation in the presence of suboptimal concentrations of concanavalin A.
INTRODUCTION It has been proposed that early during mitogen stimulation of lymphocytes are related to changes in the intracellular concentration of various ions. The ions that have been implicated in such events include Ca2+ (1, 2) and K+ (3-5). Recently it was reported that Mn2+, a putative Ca2+antagonist, inhibited an early event during stimulation of lymphocytes by mitogens (6,7). In addition zinc ions alone have been shown to be mitogenic for both human (8, 9), hamster (lo), and guinea pig (10) lymphoid cells. Another ion which has been shown to have biologic activity in a variety of systems is Li+. In fact this ion is widely utilized pharmacologically in the field of psychiatry for the treatment of manic depressive states (1 1- 13). In addition to an effect on behavior, a variety of side effects of Li+ have been noted in both man and experimental animals (12, 13). These include obesity, muscle weakness, and alterations in the immune system and lymphoid cells. These later findings include involution of the thymus of both normal and adrenylectomized mice (14), concentration of in vivo administered Li+ in the cortex of the thymus (15), lymphopenia (16, 17), and the appearance of antinuclear antibodies in a significant percentage of treated patients compared to age-matched controls (18). It is also interesting to note that in the latter study, Li+ treatment of a patient with an ’ This investigation was supported by National Institutes of Health Lymphocyte Biology AI-11851.
Grant
113 0008-8749/79/030113-10$02.00/O Copyright 0 1979 by Academic Press. Inc. All rights of reproductionin any form reserved.
114
DAVID
A. HART
autoimmune disease, systemic lupus erythematous (SLE), led to an exacerbation of the disease (18). The pharmacotherapeutic mechanisms of Li+ are still unclear. This ion has been reported to modulate the activity of a large number of enzymes (19) as well as have an effect on the distribution of other ions such as Mg2+, Ca2+, and Na+ (19). A considerable literature has accumulated which has led to the idea that the important systems that are modulated by Li+ are related to either cyclic nucleotide metabolism (20, 21) or membrane ATPases (22, 23) possibly by interfering with Mg2+ (21) or K+-dependent events (23). These latter postulated sites of action of Li+, coupled with the above-described effects of LiCl on lymphoid cells, raised the possibility that Li+ could exert a direct effect on lymphocyte activation or function. In the present report evidence is presented that LiCl can modulate serum-free stimulation of hamster lymphoid cells by the T-cell mitogen, concanavalin A. MATERIALS
AND METHODS
Materials. Concanavalin A (Con A) was obtained from Sigma Chemical Company. The salts NaCl, KCl, CaCl,, MgC12,and LiCl were all reagent grade and were obtained from Mallinkrodt Inc. Stock solutions of salts, 1 M, were prepared in glass distilled water and sterilized by filtration through sterile 0.22~pm filters (Millipore Corp.). Tissue culture medium RPMI-1640 containing penicillin and streptomycin was obtained from Associated Biomedic Systems. Tritiated thymidine ([3H]TdR), 2-5 Ci/mM, was obtained from Amersham-Searle Corporation. Cell cultures. Lymphoid tissues (spleen, lymph nodes, and thymuses) were removed from 7- to lZweek-old female MHA hamsters (Charles River Lakeview Hamster Colony) and single-cell suspension prepared. Viability was determined by the trypan blue exclusion test. The cells, 5 x lo6 viable cells in 1 ml RPMI-1640, were cultured under serum-free conditions for 72 hr in a humid atmosphere of air-lo% CO, as described previously (24). Twenty-four hours prior to harvest, 0.5-1.0 $Ji[3H]TdR was added to each culture. The cultures were harvested on glass filters and incorporated [3H]TdR was determined as previously described (24). All assays were performed in triplicate and the variation from the mean incorporation was less than 10% in all reported experiments. Addition of ions. Unless otherwise indicated in individual experiments, appropriate concentrations of salts were added 1 hr prior to the mitogen. In multiple ion experiments, the LiCl was added 2-3 min after the first salt(s) was added to the cultures. RESULTS Effect of LiCl on Con-A Stimulation of Thymocytes Addition of LiCl to unstimulated cultures of thymocytes did not alter [3H]TdR incorporation over the concentration range of I-25 mM LiCl. However addition of LiCl to cultures of thymocytes stimulated with 0.1-2.0 pg Con A led to a dose dependent inhibition of this mitogenic response (Fig. 1). The inhibition observed was dependent on both the concentration of LiCl and the concentration of Con A added to the cultures. That is, at low suboptimal concentrations of Con A, addition
Li AND LYMPHOCYTE
115
STIMULATION
CcnA CONCENTRATION
(pg/ml)
FIG. 1. Modulation by LiCl or Con A stimulation of thymocytes. MHA thymocytes were stimulated with the indicated concentrations of Con A in RPMI-1640 alone (0) or in the presence of 1 mM LiCl (0), 5 m&f LiCl (A), 10 n&f LiCl (A), or 25 mkf LiCl (e). The indicated values represent the mean incorporation of [3H]TdR into triplicate assays. The variation from the mean incorporation was less then 10%.
of LiCl to the cultures led to an enhanced incorporation of [3H]TdR while the same concentrations of LiCl inhibited optimal stimulation by Con A (Fig. 1). Experiments utilizing Con A concentrations of 0.001-0.25 pg, indicated that addition of LiCl(5- 10 m&f) to cultures treated with submitogenic doses of Con A did not lead to stimulation of [3H]TdR incorporation (Fig. 2). Thus LiCl enhanced suboptimal stimulation by Con A but did not permit stimulation by submitogenic stimuli. The finding that stimulation by suboptimal concentrations of Con A was enhanced by LiCl, as well as trypan blue exclusion test data, indicated that the concentrations of LiCl tested were not toxic for the cells.
-0
o.ml
001
O.C6
Con A CONCENTRATION
025
03 (pghl
1
FIG. 2. The effect of LiCl on suboptimal Con A stimulation of MHA thymocytes. Cells were stimulated with the indicated concentrations of Con A in RPMI-1640 alone (0) or in the presence of 5 mM LiCl (0), or 10mM LiCl (A). The indicated values represent the net mean incorporation of 13H]TdR into triplicate assays. The variation from the mean incorporation was less than 10%.
116
DAVID A. HART
+24 TIME
OF ADDlTlON
OF LiCl
(hrsl
FIG. 3. The effect of time of addition on LiCl inhibition of Con A stimulation of MHA thymocytes. Cells were stimulated with 1 pg Con A (0,O) or 2 pg Con A (&A) in RPMI-1640 (control). At the indicated times 10 mM LiCl (.,A) or 25 mM LiCl (0,A) was added to appropriate cultures. The time of addition of the Con A was considered T = 0. The indicated values represent the mean incorporation of [3H]TdR into triplicate assays. The variation from the mean incorporation was less than 10%.
This latter conclusion was also supported by time of addition experiments. Lithium chloride was added to cultures of thymocytes at various times after stimulation with 1 and 2 pug Con A. As the data depicted in Fig. 3 indicate, concentrations of LiCl which were strongly inhibitory when added at the initiation of the culture, rapidly lost their ability to inhibit the response to Con A. The most dramatic loss in inhibition occurred within the first 24 hr of culture. When LiCl was added after 48 hr of culture, little or no inhibition of the Con A response was observed (Fig. 3). This latter finding eliminated the trivial explanation that the inhibition observed was due to LiCl interference with [3H]TdR transport and incorporation into the cells. Effect of LiCl on Con A Stimulation
of Lymph Node Cells and Splenocytes
Similar to results obtained with thymocytes, addition of LiCl to cultures of lymph node cells (Fig. 4) or splenocytes (data not shown), stimulated with suboptimal concentrations of Con A, led to an enhancement of [3H]TdR incorporation. However, in contrast to results obtained with thymocytes, stimulation of lymph node cells by optimal concentrations of Con A was not strongly inhibited by 10 mM LiCl (Fig. 4). When the concentration of LiCl was raised to 25 rnM, extensive inhibition of the Con A response was observed. These results indicated that stimulation of lymph node cells may be more refractory to the effects of LiCl than thymocyte populations. Also in contrast to the findings obtained with thymocytes, addition of 5- 10 mM LiCl to unstimulated cultures of lymph node cells, and to a lessor extent splenocytes, led to a two- to six-fold stimulation of [3H]TdR incorporation. Specificity
of the Effect of LiCl on Lymphocyte
Stimulation
To determine whether the observed effects of LiCl on Con A stimulation of lymphoid cells, particularly thymocytes, was specific for this ion, several other ions
Li AND LYMPHOCYTE
ConA
STIMULATION
CONCENTRATION
117
(pg/ml)
FIG. 4. Modulation by LiCl of Con A stimulation of MHA lymph node cells. Cells were cultured in the presence of the indicated concentrations of Con A in RPMI-1640 alone (@) or after addition of 10 mM LiCl (O), or 25 rnkf LiCl (A). The indicated values represent the mean incorporation of [3H]TdR into triplicate assays. The variation from the mean incorporation was less than 10%.
were also tested. Previously it has been shown that 50 @4 ZnCl, (10) or 50- 100pM MnCl, (6) inhibits Con A responses from all tissues tested over the entire dose range of Con A. Ions tested in the present study which had no inhibitory effect on Con A stimulation of thymocytes were NaCl(25 mM), KC1 (25 mkf), MgC& (25 m&f), and CaCl, (1.6 mM). From these results it was concluded that the inhibitory effect of LiCl on Con A stimulation of thymocytes was relatively specific for this ion and was not due to an osmotic effect. While the ions tested did not inhibit Con A stimulation of lymphoid cells, some of those tested, particularly Mg2+, did enhance stimulation by suboptimal concentrations of Con A (discussed in more detail below). Reversal of the Inhibitory Effect of LiCl by Other Ions It is believed that Li+ exerts some of its biological effects through antagonism with other ions in enzyme systems. This ion has been postulated to interfere with the activity of adenyl cyclase via an antagonism with Mg2+(21) or to modify the activity of NaK-ATPases through antagonism with K+ (23). To determine whether Li+ was exerting an inhibitory effect on Con A stimulation of thymocytes by displacing one or more of these other ions from a system essential for activation of thymocytes, attempts were made to reverse the inhibitory effect of Li+ on Con A stimulation with Mg2+, Ca2+, Mg2+ + Ca2+, Na+, K+, and K+ + Mg2+. In contrast to LiCI, MgC12(5-25 n&f) did not increase the [3H]TdR incorporated into unstimulated cultures of splenocytes or lymph node cells. In addition, the inhibition of Con A stimulation by LiCl was not drastically altered by the presence or absence of 25 rn44 MgCl, (Fig. 5) although there was some reversal of the inhibition induced by 10 mk! LiCl noted in the presence of 25 mkf MgC12.However, when compared to the control (Fig. 5a), addition of 25 mM MgCi, (Fig. 5b) to cultures of thymocytes stimulated with Con A led to an enhancement of [3H]TdR incorporation over the entire dose range of Con A; but in particular at the 0.1 wg Con A level where a 15-fold enhancement of [3H]TdR incorporation was observed.
118
DAVID A. HART
ConA UJNCENlRATlON
(pghnl)
FIG. 5. The effect of MgCl, on LiCl modulation of Con A stimulation. Thymocytes were cultured in RPMI-1640(A) or in RPMI-1640 + 25 mb4 MgC& (B). The cells were then stimulated with the indicated concentrations of Con A with no further additions (0) or after addition of either 10 mM LiCl(0) or 25 mM LiCl (A). The indicated values represent the mean incorporation of [3H]TdR into triplicate assays. The variation from the mean incorporation was less than 10%.
Similar results were obtained from cultures of lymph node cells or splenocytes (data not shown). It should be noted that the enhancement of suboptimal stimulation of r3H]TdR incorporation observed when MgC& or LiCl was added to cultures was further enhanced when 10 mk! LiCl + 25 rnkf MgC& was added but there was no induction of stimulation by submitogenic doses of Con A (Fig. 6). At the 0.1 ,ug Con A level, the [3H]TdR incorporated in the presence of Mg + Li was 253-fold enhanced over that incorporated with no additional Mg or Li. Therefore rather than an antagonism in this system there appears to be an additive effect of the two ions. Results similar to those described above for 25 mM MgC& (Fig. 5) were obtained when attempts were made to reverse LiCl inhibition with either 1.6 m&f Ca2+or 25 mA4 MgC12 + 1.6 mM CaCl, (data not shown). That is suboptimal Con A stimulation was enhanced in the presence of these ions but LiCl-induced inhibition was nor reversed. From these three sets of experiments it would appear that LP was not exerting an inhibitory/enhancement effect on Con A stimulation via antagonism with either Mg2+ or Ca2+. Lithium has also been proposed to be an antagonist of K+ ions (23). This latter ion has been postulated to be important in lymphocyte stimulation by several investigators (3-5). Rapid alterations in the concentration of this ion have been reported to occur after mitogen stimulation (3-5). This is believed to occur via
Li AND LYMPHOCYTE
STIMULATION
ConA CONCENTRATION
119
(pg/ml)
FIG. 6. Potential of suboptimal Con A stimulation by LiCl and M&l,. Thymocytes were stimulated with the indicated suboptimal concentrations of ConA in RPMI-1640 alone (0) or after addition of 10mM LiCl(O), 25 nGt4MgCI, (A), or 10n&f LiCl + 25 mM MgCl, (A). The indicated values represent the net mean incorporation of [3H]TdR into triplicate assays. The variation from the mean was less than 10%.
modification of the activity of a membrane NaK-ATPase (3). Inhibition of this ATPase with the specific inhibitor ouabain blocks early stimulation events (3, 25, 26). Therefore experiments were preformed to evaluate the effect of other monovalent cations on thymocyte stimulation. Preliminary experiments indicated that addition of 25 mM NaCl to cultures of thymocytes stimulated with Con A (0.1-2.0 pg) had no effect on either stimulation or LiCl-induced inhibition (data not shown). Additional attempts were then made to reverse LiCl inhibition of Con A stimulation with KCl. As seen in Fig. 7, addition of IO-25 mM KC1 to Con A stimulated cultures had no effect on stimulation but addition of these concentrations of KC1 to LiCl-inhibited cultures led to a significant reversal of the observed inhibition. The reversal of inhibition was most notable at the l-pg Con A level. At this level of Con A there was a dose-dependent reversal by 10and 25 mM KC1 of the inhibition induced by 25 n&f LiCl (Fig. 7). In the control cultures, stimulation by l-pg Con A was inhibited 70% by 25 mM LiCl while in cultures with 10 and 25 mM KC1 this concentration of LiCl inhibited the l-Fg Con A response by only 30% and 8%, respectively. As Li+ has been postulated to exert biological effects via antagonism with Mg2+ or K+, attempts were then made to reverse LiCl inhibition of Con A stimulation with a combination of K+ + Mg2+. When such experiments were performed with 12.5 mM MgCl, + 12.5 mM KC1 or 20 mM MgC& + 20 mM KCl, there was no dramatic reversal of LiCl inhibition (data not shown). The inhibition observed in the presence of both Mg2+ and K+ was not significantly different from that seen in the presence of K+ alone (Fig. 7). This finding indicated that Li+ was not exerting an effect via antagonism with two separate essential systems, one Mg2+ dependent and the other K+ dependent.
120
DAVID A. HART
120 / / 60 ,I’.,’ 60 x,p ::40 ./?/ 0
I. .r-.‘\, ,’,Ii ,.I ‘.’
0.1 1.0 2.0 CatA CONCENTRATloN (#nil
FIG. 7. The effect of KC1 on LiCl modulation of Con A stimulation. Thymocytes were cultured in RPMI-1640, RPMI-1640 + 10 mM KCI, or RPMI-1640 + 25 nGt4KCl. The cells were then stimulated with the indicated concentrations of Con A with no further additions (0) or after addition of 10mM LiCl (0), or 25 m44 LiCl(A). The indicated values represent the mean incorporationof [3H]TdRinto triplicate assays. The variation from the mean was less than 10%.
DISCUSSION The results presented in this study demonstrate that LiCl can have a direct effect on lymphocyte stimulation. The points that may be concluded from the data include the following: (i) LiCl potentiates suboptimal Con A stimulation; (ii) LiCl inhibits optimal and supraoptimal Con A stimulation; (iii) LiCl acts to modify early stimulation events; (iv) the action of LiCl is unique to lithium; and (v) thymocytes exhibit a sensitivity to the effects of LiCl not seen with lymph node cells or splenocytes. The mechanism by which Li+ exerts a biological effect in most systems is not well understood. However, the possibilities that have been raised include alteration of cyclic nucleotide metabolism (20,21) or modification of membrane enzymes such as membrane NaK-ATPases (22, 23). Both of these possibilities are relevant to lymphocyte stimulation since both cyclic nucleotides [reviewed in (27, 28)] and changes in specific ion flux (l-5) have been implicated as early activation events.
Li AND LYMPHOCYTE
STIMULATION
121
The results obtained in the present study would tend to support the latter hypothesis rather than the former cyclic nucleotide hypothesis. This conclusion is based on the finding that LiCl-induced inhibition of stimulation was partially reversed by K+ (Fig. 7) but not Mg2+ (Fig. 5) and the fact that Li+ + Mg2+ yielded additive enhancement of suboptimal stimulation (Fig. 6). As Li+ has been postulated to alter cyclase activity via antagonism with Mg2+ (21), it is doubtful that Li+ modulates lymphocyte activation via this mechanism since Li+ and Mg2+ appeared to act in a positive fashion in the present report. The hypothesis that Li+ acts by altering specific ion flux is very attractive for a variety of reasons. It has been proposed, and supported by experimental observations, that interaction of lectins such as Con A with lymphoid cells leads to rapid changes in ion flux across the plasma membrane. The two ions which have been implicated are K+ (3-5) and Ca2+(1,2). Modification of the flux of the former ion, K+, is thought to be mediated via membrane-bound NaK-dependent ATPases (4) and the importance of this enzyme activity has been demonstrated with the specific inhibitor, ouabain (3, 25, 26). Addition of this inhibitor to cultures of lymphocytes stimulated with lectins leads to inhibition of early activation events (25). In addition, Li+ has been shown to activate some NaK-dependent ATPases, even in the presence of saturating concentrations of K+ (23). Apparently Li+ displaces K+ from some sites on the enzyme which regulate the activity of the enzyme. In the present study K+ ions were the only effective cations which modified the inhibitory phase of the Li+ effect on Con A stimulation. If indeed specific ion flux is necessary for mitogenic stimulation then suboptimal stimulation could be limited by the ability of the stimulant to induce a primary mitogenic event and then modify rate limiting systems. This statement is based on the inability of Li+ to induce stimulation by itself or to induce mitogenesis by submitogenic concentrations of Con A, and assuming the enzyme is active in the membrane of the unstimulated cell, changes in the activity of the enzyme alone would not be a primary mitogenic signal. However, the enhancement by ions (LiCl + MgCl,) of stimulation by suboptimal concentrations of Con A, to levels comparable to that obtained with optimal concentrations of Con A, indicates that possibly the additional Con A is not inducing a primary mitogenic signal but rather is modifying rate-limiting steps in the activation process. The enhancing effect of Lit on suboptimal Con A stimulation could then result from activation of such a rate-limiting event. Therefore addition of LiCl to optimally stimulated cultures led to inhibition because of “over-activation” of the appropriate activities. A second issue raised by the data presented is related to the apparent unique sensitivity to LiCl of thymocyte stimulation. This data provides evidence that the previously reported involution of the thymus following in vivo administration of LiCl could be due to a direct effect of Li+ on thymic tissue (14). This conclusion is also supported by the work of Nelson ef al. (15) who reported that the thymic cortex concentrates in vivo-administered Li+. These observations raise the possibility, first proposed by Perez-Cruet and Dancey (14), that chronic administration of LiCl could lead to altered immune responses. The accumulated data, presented in this report and previously discussed under Introduction, strongly suggest that one ofthe immune systems which could be modified by LiCl is the suppressor T-cell system. Reports from several laboratories have indicated that suppressor T cells are short lived [reviewed in (29)], and a decline in suppressor-cell function has been
122
DAVID A. HART
postulated to accompany age and thymic involution (30-32). Such a hypothesis would predict that the progressive lymphopenia (16, 17) observed during chronic Li+ administration is due to a decline in a T-cell population. Further analysis of these hypotheses for the effect of Li+ on immune functions is currently in progress. ACKNOWLEDGMENTS The author wishes to thank Janet Frazier for excellent technical assistance during the course of this investigation, Daisi Marcoulides for secretarial assistance in the preparation of the manuscript, and Drs. Joan Stein-Streilein and R. Jerrold Fulton for their review of the manuscript.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
Whitney, R., and Sutherland, R. M., Cell Zmmunol. 5, 137, 1972. Freedman, M. H., Raff, M. C., and Gomperts, B., Nature 255, 378, 1975. Quastel, M. S, Dow, D. S., and Kaplan, J. G. Proc. Leucocyre Cult. Conf. 5, 97, 1970. Averdunk, R., and Lauf, P. K., Exp, Cell Res. 93, 331, 1975. Segel, G. B., and Lichtman, M. A., J. Clin. Invest. 58, 1358, 1976. Hart, D. A., Exp. Cell Res. 113, 139, 1978. Hart, D. A., Fed. Proc. 37, 1550, 1978. Kirchner, H., and Rilhl, H., Exp, Cell Res. 61, 229, 1970. Berger, N. A., and Skinner, A. M., J. Cell Biol. 61, 45, 1974. Hart, D. A., Zfect. Zmmunol. 19, 457, 1978. Kupfer, D. J., (Ed.), “Lithium and Psychiatry Journal Articles.” Medical Examination Publ. Co., Flushing, N.Y., 1971. Johnson, F. M., and Johnson, S., (Eds.), “Lithium in Medical Practice.” University Park Press, Baltimore, 1977. Jefferson, J. W., and Greist, J. H. (Eds.), “Primer of Lithium Therapy.” Williams & Wilkins, Baltimore, 1977. Perez-Cruet, J., and Dancey, J. T., Experientiu 33, 646, 1977. Nelson, S. C., Herman, M. M., Bensch, K. G., Sher, R., and Barchas, J. D., Exp. Mol. Pathol. 25, 38, 1976. Perez-Cruet, J., Dancey, J. T., and Waite, J., In “Lithium and Medical Practice.” (F. N. Johnson and S. Johnson Eds.), p. 271, University Park Press, Baltimore, 1977. Radomski, J., Fuyat, H. N., Nelson, A. A., and Smith, P. K., J. Pharmacol. 100, 429, 1950. Presley, A. P., Kahn, A., and Williamson, N., bit. Med. J. 2(6030), 280, 1976. Birch, N. J., In “Lithium in Medical Practice.” (F. N. Johnson and S. Johnson, Eds.), p.89, University Park Press, Baltimore, 1977. Geisler, A., Klysner, R., and Thams, P. In “Lithium and Medical Practice.” (F. N. Johnson and S. Johnson, Eds.), p. 159, University Park Press, Baltimore, 1977. Wang, Y-C., Pandey, G. N., Mendels, J., and Frazer, A., Biochem Pharm. 23, 845, 1974. Glen, A. I. M. In “Lithium in Medical Practice.” (F. N. Johnson and S. Johnson, Eds.), p. 183, University Park Press, Baltimore, 1977. Robinson, J. D., Biochim, Biophys, Acta 413, 459, 1975. Streilein, J. S., and Hart, D. A., Znfec. Zmmunol. 14, 463, 1976. Quastel, M. R., and Kaplan, J. G. Nature (London) 219, 198, 1968. Negendank, W. G., and Collier, C. R., Exp. Cell Res. 101, 31, 1976. Wedner, H. J., and Parker, C. W., Prog. Allergy 20, 195, 1976. Oppenheim, J. J., and Rosenstreich, D. L., Prog. Allergy 20, 65, 1976. Moller, G. (Ed.), Transplant. Rev. 26, 1975. Okumura, K., and Tada, T., J. Zmmunol. 106, 1019, 1971. Kerbel, R. S., and Eidinger, D., Eur. J. Zmmunol. 2, 114, 1972. Allision, A. C., Denman, A. M., and Barnes, R. D., Lancer 1, 135, 1971.
