CELLULAR IMMUNOLOGY 89, 322-330 (1984)

Modulatory Effect of Zinc on the Proliferative Response of Murine Spleen Cells to Polyclonal T Cell Mitogens’

Itis MALAV~ AND ISAAC RON~~N BENA~M

Department of Experimental Medicine, Instituto Venezolano de Investigaciones Cientijicas (I. V.I.C.), Apartado 1827, Caracas 1010 A. Venezuela

Received March 7, 1984; accepted May 29, 1984

To study the effect of zinc on the proliferative response to polyclonal T cell mitogens, spleen cells from C57BL/6 mice were cultured with or without ZnC12 and stimulated with graded doses of concanavalin A or phytohemagglutinin. Addition of IO-’ M ZnClr inhibited proliferation whereas lo-’ to 10” M ZnClr did not modify the response to suboptimal doses of mitogen but increased DNA synthesis in cultures stimulated with high doses of mitogen (10 or 20 &ml of concanavalin A and 10 or 25 &ml of phytohemagglutinin) which are supraoptimal for C57BL/6 mice, and inhibited proliferation in cultures of spleen cells from animals of this strain, low responder to T cell mitogens. In contrast, supplementation with ZnClz did not enhance the response to mitogen of spleen cells from high responder BALB/c mice. The enhancing effects of ZnCl, on the proliferative response of C57BL/6 cells were not observed following depletion of adherent cells or in cultures supplemented with 5 X 10m5 A4 2-mercaptoethanol, both conditions capable of abrogating the inhibitory effect of high mitogen doses on the response of C57BL/6 cells. 0 1984 Academic press, h.

INTRODUCTION

Zinc participates in various events leading to lymphocyte proliferation. It is a cofactor of several enzymatic systems essential for DNA and RNA synthesis (1, 2) metabolic processes which are significantly impaired in zinc deficiency (3, 4). It has also been shown that zinc is an essential requirement of the proliferative response to T cell mitogens (5, 6), and acts as a cofactor of carboxypeptidase (7), a major component of Interleukin 1 (8). In addition, when added directly to the culture system, zinc has mitogenic activity on a population of human lymphocytes (9, lo), enhances the response to phytohemagglutinin (PHA) and concanavalin A (Con A) of peripheral lymphocytes from young but not old human donors (1 I), and is both mitogenic and a potentiator of the proliferative response to lipopolysaccharide (LPS) of hamster lymph node cells and guinea pig spleen and lymph node cells (12).

Differences in the level of the proliferative response to T cell mitogens which appear to be under autosomal polygenic control have been observed among inbred mouse strains (13, 14). After stimulation with optimal doses of mitogen, spleen cells from low responder mice frequently show lower DNA synthesis than spleen cells from high responder mice. The main difference in the proliferative response of low

’ This work was partially supported by Grant NUT-2 from the “Consejo National de Investigaciones Cientificas y Tecnologicas” CONICIT, Venezuela.

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0008-8749/84 $3.00 Copyright Q 1984 by Academic press, Inc. AU rights of reproduction in any form trscrved.

MODULATION OF LYMPHOCYTE PROLIFERATION BY ZINC 323

responder and high responder strain mice is observed however in cultures stimulated with high doses of mitogen, since cells from low responder animals have marked susceptibility to their inhibitory effect (15). Depletion of adherent cells (AC) results in significant increase of DNA synthesis in cultures of low responder spleen cells stimulated with high doses of mitogen, indicating that the differences in the magnitude of the proliferative activity between low and high responder strains are related to differences in the regulation of the response to supraoptimal doses of mitogen (15) which is exerted by adherent suppressor cells (16- 18).

Interestingly, supplementation with zinc in vivo enhances the mitogenic response of peripheral blood lymphocytes from subjects in which low proliferative responses were observed before zinc administration (19) and of spleen cells from Swiss Webster mice (20). In view of the above, we have studied the effect of the addition of ZnClz to the culture system on the proliferative response to various doses of mitogen of spleen cells from low responder C57BL/6 (B6) and high responder BALB/c (C) mice in several culture conditions.

MATERIAL AND METHODS Experimental animals. Inbred male B6 and C mice, 7 to 9 weeks old, were

obtained from the Jackson Laboratory, Bar Harbor, Maine or bred at IVIC. Cell suspensions. Spleen cells pooled from four to six mice were used in each

experiment. Cell suspensions were prepared in culture medium RPM1 1640 by pressing the spleens between the frosted ends of microscope slides and then were filtered through stainless-steel wire screen mesh 200, counted, and adjusted to a concentration of 5 X IO6 cells per ml.

Depletion of adherent cells. Cells adherent to plastic surfaces were removed by plating lo8 spleen cells in 10 ml of culture medium supplemented with 10% fetal calf serum (FCS) (Grand Island Biological Lab., Grand Island, N.Y.) twice for 1 hr each at 37°C on 120-mm plastic Petri dishes.

Culture procedure. Cultures were carried out in RPM1 1640 medium, supplemented with 2 mML-glutamine, antibiotics, 5% heat-inactivated FCS (all from Grand Island Biological Lab., Grand Island, N.Y.), and 5 mM HEPES buffer. Aliquots containing 5 X 10’ spleen cells in 0.1 ml volume were delivered into the wells of flat-bottom microculture trays (Linbro-IS-FB-96-TC, International Scientific Instruments, Cary, Ill.). PHA (Reagent grade HA1 5, Wellcome Research Laboratories, Beckenham, England), and Con A (Sigma Chemical Co., St. Louis, MO.) were diluted to the desired concentration in culture medium and 0.1 ml added to each well. Unless otherwise stated, cultures were incubated for 72 hr at 37°C in humid 5% CO* in air, pulsed with 0.5 &i of [3H]thymidine (specific activity 2 Ci/mmol, Amersham, Searle) for the final 16 hr of incubation, and collected by suction on glass-fiber filters using a multiple automated sample harvester (MASH II, Microbiological Associates Inc., Bethesda, Md.). Radioactivity was measured in a liquid scintillation spectrometer and the results expressed as the arithmetic mean of triplicate cultures.

Sets of parallel cultures were supplemented with 25 ~1 of ZnClz (Sigma Chem. Co., St. Louis, MO.) solution at a final concentration ranging from 10m4 to 10m6 M or with 25 ~1 of physiologic saline solution.

RESULTS Addition of 10e4 M ZnClz to the cultures markedly inhibited the proliferative

response of B6 spleen cells since this concentration of the metal salt is toxic for

324 MALAW? AND RON&N BENAiM

lymphoid cells. Addition of ZnCls in concentrations ranging from lop5 to 10m6 M enhanced the response to optimal and supraoptimal doses of Con A but did not affect the response to suboptimal doses of the mitogen. The maximal enhancement of DNA synthesis in the presence of ZnClz was observed in cultures of B6 spleen cells stimulated with high doses of Con A (10 and 20 &ml). Although in control cultures without ZnClz these mitogen doses induced responses significantly lower than that observed after stimulation with an optimal dose, in the presence of the metal salt the response to 10 and 20 &ml of Con A was markedly increased (Fig. 1).

Since supplementation of the cultures with ZnC12 interfered with the inhibitory effect of high doses of mitogen on B6 spleen cells, and changed their dose-response curve to a pattern resembling that observed in cultures of high responder C spleen cells, it was interesting to compare the effect of the addition of ZnClz on the response of low responder B6 and high responder C cells. As shown in Fig. 2A, ZnCls markedly enhanced the response of B6 spleen cells to doses of Con A which are supraoptimal for this strain and inhibit DNA synthesis in control B6 cultures without ZnC&. In contrast, the response of C spleen cells was not affected at all the mitogen dose range tested. In some experiments, as the one shown in Fig. 2B, addition of ZnCl* moderately enhanced the response of C spleen cells to the highest dose of Con A used (20 &ml). However, the enhancing effect was much more striking in cultures of B6 spleen cells in which, in the same experiment, the response to 20 &ml of Con A increased more than 20 times.

It has been shown previously that depletion of AC enhances the response to high doses of PHA and Con A of low responder B6 spleen cells (15). As described above,

6% b lb do Con A dose (pg /ml )

FIG. 1. Effect of the addition of ZnClz on the proliferative response to Con A of spleen cells from C57BL/6 mice. (0 - 0) Control cultures without ZnC&; (X - - - X) cultures supplemented with lO-4 M ZnC12; (0 - - - 0) cultures supplemented with 10S5 M ZnC12; (A - - - A) cultures supplemented with 5 X 10e6 M ZnCl,; (A - - - A) cultures supplemented with lo-’ M ZnCls.

MODULATION OF LYMPHOCYTE PROLIFERATION BY ZINC 325

015 4 lb 1 20

Con A dose (pg/ml 1 Con A dose (pg/ml)

FIG. 2. E&t of the addition of ZnCl* on the proliferative response to Con A of spleen cells from high responder BALB/C (open symbols) and low responder C57BL/6 mice (closed symbols). (-) Control cultures without ZnCl*; (- - -) cultures supplemented with 5 X 10m6 M ZnClz; (-. -.-) cultures supplemented with 10e6 M ZnClz .

addition of ZnClz to cultures of complete spleen cells from B6 mice markedly increased the proliferative response to a high, moderate supraoptimal dose of Con A whereas the addition of a similar concentration of the metal salt did not modify the dose-response curve in cultures of AC-depleted B6 spleen cells, only causing moderate enhancement of the response to 20 pg/ml of Con A (Fig. 3).

Supplementation of the culture medium with 5 X lo-’ A4 2-mercaptoethanol (2-ME) increased the response of B6 spleen cells to suboptimal and optimal Con A doses and diminished their sensitivity to the inhibitory effect of high doses of Con A (10 and 20 &ml), also increasing DNA synthesis in cultures stimulated with these supraoptimal doses of mitogen. Although addition of ZnClz increased the proliferative response to high doses of mitogen in cultures without 2-ME, the metal salt did not enhance lymphocyte proliferation in cultures supplemented with 5 X lo-’ M 2-ME (Fig. 4). Similar results were obtained in cultures stimulated with PHA (Fig. 5). It could also be observed in these experiments that the amplifying effect of 2-ME was more marked than that of ZnClz.

In order to mediate enhancing effects and the typical changes in the dose- response curve of B6 spleen cells, ZnCl* should be present at the beginning of the culture. Thus, addition of the metal salt 15 hr after the initiation of mitogen stimulation produced less enhancement than its earlier addition. ZnClz did not affect the response to Con A at all the mitogen dose range tested when added 24 hr after the initiation of the culture (Fig. 6).

326 MALAVe AND RONtiN BENAiM

I 20

Con A dose (~q/mlI

FIG. 3. Effect of the addition of ZnClz on the proliferative response to Con A of whole spleen cells (closed symbols) or adherent cellsdepleted spleen cells (open symbols) from C57BL/6 mice. (-) Control cultures without ZnCb; (- - - ) cultures supplemented with lo+ M ZnC&.

j&5 4 lb I don A dose (pg /ml 1

20

FIG. 4. Effect of the addition of Z&l2 on the proliferative response to Con A of spleen cells from C57BL/6 mice cultured in the presence (open symbols) or in the absence (closed symbols) of 5 X 10d5 M 2-mercaptoethanol. (-) Control cultures without ZnQ; (- - -) cultures supplemented with low5 A4 ZnC& .

MODULATION OF LYMPHOCYTE PROLIFERATION BY ZINC 321

J%25 IO I 1 1 1

25 PHA dose (pg/ml)

FIG. 5. Effect of the addition of ZnQ on the proliferative response to PHA of spleen cells from C57BL/6 mice cultured in the presence (open symbols) or in the absence (closed symbols) of 5 X 10m5 M 2-mercaptoethanol. (-) Control cultures without ZnQ; (- - -) cultures supplemented with IO4 M ZnQ.

DISCUSSION

It has been shown here that addition of zinc to the cultures enhanced the proliferative response of spleen cells from B6 mice characterized by their genetically determined low responsiveness to Con A and PHA. The enhancement of the proliferative response of B6 spleen cells was mainly observed in cultures stimulated with high doses of mitogen which inhibited DNA synthesis in control cultures without ZnC&, but did not occur in cultures stimulated with suboptimal doses of Con A and PHA. The enhancing effect of ZnClz was significantly less or was not evident when similar concentrations of the metal salt were added to cultures of spleen cells from high responder C mice.

Since the enhancement of the lymphoproliferative response by ZnCl, was mitogen dose dependent, physical phenomena due to the direct addition of the heavy metal to the cultures which could mimic increased [3H]thymidine incorporation into DNA can be discarded. This possibility is yet less likely in view of the enhancement of the antibody response following in vitro stimulation with T-dependent and T-independent antigens observed in cultures supplemented with ZnClz (21).

It has been shown previously that the proliferative response to mitogens is modulated by AC. This cell population either increases or decreases mitogen- induced DNA synthesis according to their proportion in the culture system (22, 23), the degree of macrophage activation (18, 24), but also in a dose-dependent fashion (18). High AClymphocyte ratios (22, 23) and/or activation of macrophages (18, 24) result in depressed responses. On the other hand, the AC population contributes to the inhibition of the proliferative response in cultures stimulated with supraoptimal doses of mitogen (16-18), whereas the response to low doses of mitogen shows high AC dependency (15, 25). In a previous work it was shown that

328 MALAVE AND RONDON BENAiM

%k 5 lb 1 20

Con A dose (pg/ml) Con A dose (pglrnl) Con A dose l,ug/ml)

FIG. 6. Effect of the addition of ZnClr at different times of culture on the proliferative response to Con A of spleen cells from C57BL/6 mice. ( -) Control cultures without ZnC12; (- - -) cultures supplemented with 10m6 M ZnClz. (A) ZnQ added 30 min before mitogen; (B) ZnClt added immediately after mitogen; (C) ZnCll added 2 hr after mitogen; (D) ZnC& added 3.5 hr after mitogen; (E) ZnClz added 15 hr after mitogen; (F) ZnClr added 24 hr after mitogen.

the proliferative response to high doses of mitogen of spleen cells from low responder B6 mice increases after depletion of AC (15). In fact, AC-depleted B6 spleen cell cultures lose sensitivity to the inhibitory effect of supraoptimal doses of Con A and show a dose-response curve similar to that of high responder C spleen cells (15). Since addition of zinc does not markedly modify the response of AC-depleted B6 spleen cells it could be thought that the ion counteracts the suppressor function of AC either affecting them directly, interfering with their soluble mediators of suppression (22, 23, 26), or favoring the secretion and/or activity of AC stimulatory factors (27). It could also be possible that the cell population responsive to high doses of mitogen differs from that responsive to low and optimal mitogen doses. In the presence of AC, zinc could contribute to the activation of cells capable of responding to high doses of mitogen. It should be mentioned in this regard that zinc acts as a polyclonal lymphocyte activator ( 12, 28). This ion has direct mitogenic effects on some populations of human peripheral lymphocytes (9, lo), and of hamster and guinea pig lymphoid cells (12), and in the present work a moderate but inconstant mitogenic effect was observed in some experiments in control cultures without lectins supplemented with ZnClz. On the other hand, zinc is a

MODULATION OF LYMPHOCYTE PROLIFERATION BY ZINC 329

cofactor of the active form of the thymic serum factor (29) and could promote the differentiation of some cell populations and render them responsive to mitogens. In fact, increased numbers of immature lymphocytes as demonstrated by high numbers of cell forming autologous rosettes have been detected in zinc deficiency (30).

Interestingly, the enhancing effects of zinc were less evident or were not apparent in cultures of B6 spleen cells supplemented with 5 X lop5 M 2-ME, in which the response to mitogens is significantly increased. It is important to notice that besides improving the response to optimal and supraoptimal doses of mitogen, 2-ME greatly enhanced the response to suboptimal doses of mitogen. Thus, 2-ME modified the mitogen dose-response relationship in a different fashion than zinc, suggesting that either agent acts through different mechanisms.

It is interesting to note that zinc did not potentiate lymphocyte proliferation when the level of the response to large doses of mitogen was high, namely, in cultures of B6 spleen cells depleted of AC or supplemented with 2-ME, and in cultures of high responder C spleen cells. These results agree with a previous work showing that supplementation with zinc in vivo only enhanced the proliferative response to T cell mitogens in individuals showing low proliferative responses prior to zinc administration (19). The results also coincide with the potentiating effects of zinc on the antibody response to in vitro antigenic stimulation which were mainly observed in cultures without 2-ME (21). It appears, therefore, that zinc enhances the proliferative activity only when the level of the response in control cultures without ZnClz is low. Addition of zinc to the cultures could either promote the activity of regulatory cells or mediators which exert amplifying effects when the response of the assay system is low, or could interfere with cells or factors contributing to the suppressor regulation of the response.

It has also been postulated that the suppressor effect of high doses of Con A depends on modifications of the cell membrane and its receptors for mitogens (3 1). Zinc modifies the number and mobility of the receptors for mitogen (32) and this fact could contribute to the modulation of the proliferative response in cultures supplemented with the metal. It cannot be discarded, however, that other cellular effects mediated by zinc, like changes in membrane-bound enzymes, in the flux of other ions and in the cytoskeleton (reviewed in 33) could modify the activation of responsive cells and/or the interaction of cells in the cultures.

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