effect of zinc on the proliferation response of human lymphocytes: mechanism of its mitogenic action

Immunopharmacology, 20 (1990) 1-10 Elsevier

IMPHAR 00501

Effect of zinc on the proliferative response of human lymphocytes" mechanism of its mitogenic action

In6s M a l a v 6 1, J a i m e R o d r i g u e z 2, Z a i d a A r a u j o 1 a n d I n g r i d R o j a s 1

l Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Cientificas (I. V.L C.), and 2Itospital Universitario de Caracas, Caracas, Venezuela

(Received 24 November 1989; accepted 31 March 1990)

Abstract: To study the effect of Zn on the proliferative response of normal human lymphocytes, ZnC12 at a final concentration of 10-4 M was added to cultures of peripheral blood mononuclear cells (PBMC) stimulated with concanavalin A (Con A) and to autologous mixed lymphocyte cultures of responder T lymphocytes and irradiated autologous non-T cells. Addition of Zn increased by about 50~0 the synthesis of DNA in cultures stimulated with either 10 or 20 #g/ml of Con A and markedly enhanced the autologous mixed lymphocyte reaction, which increased about 5-fold in the presence of Zn. In a narrow dose range, Zn induced per se the incorporation of [3H]thymidine by PBMC, with maximal effects in cultures stimulated with 10-4 M ZnC12. The percentage of cells expressing receptors for IL-2 and transferrin as assessed by immunofluorescence with the monoclonal antibodies (mAb) anti-Tac and OKT9, respectively, significantly increased when PBMC were stimulated with 10 -4 M ZnC12 alone. Maximal [3H]thymidine incorporation and maximal percentage of cells bearing those activation markers were observed on day 6 of culture. Thus, the increase in the uptake of [3H]thymidine induced by Zn is not artifactual but due to progression in the cell cycle. Incubation with the mAb anti-Tac significantly inhibited the proliferative response to Zn, indicating that this requires binding of IL-2 to its receptor. However, addition of human recombinant IL-2 did not increase [3H]thymidine incorporation by PBMC cultured in the presence of ZnC12.

Key words: Zinc; Lymphocytes; Lymphocyte proliferation; Mitogenesis; Interleukin-2 receptor; Transferrin receptor; Mono- clonal antibody

Introduction

Zinc is an essential element for cell proliferation as it part icipates in various enzymatic systems required for D N A and R N A synthesis (Parisi and

Correspondence: In6s Malav6, Centro de Medicina Experi- mental, IVIC, Aptdo. 21827, Caracas 1020A, Venezuela Abbreviations: mAb, monoelonal antibodies; SRBC, sheep red blood cells; PBMC, peripheral blood mononuclear cells; AMLR, autologous mixed lymphocyte reaction; FBS, fetal bovine serum; IL-2, interleukin 2; IL-2R, interleukin 2 receptor; TrR, transferrin receptor.

Vallie, 1970; P rasad and Oberleas, 1974). These metabolic events are significantly impaired when Z n is deficient either in vivo (Sans tead and Rinaldi, 1973; Flynn, 1984; Zanzon ico etal . , 1984) or in the presence of Z n chelators in vitro (Rubin, 1972; Williams and Loeb, 1973). It has also been shown that zinc depletion preferentially affects the proliferation of T lymphocytes (Flynn, 1984; Zanzon ico et al., 1984).

The influence of this metal on the immune response is clearly demons t ra ted by the thymus atrophy, lymphopenia and immunodepress ion

0162-3109/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

which accompany the enteropathy of the A-46 mutant of Danish Fresian cattle (Brummensted etal., 1971) and by the immunodeficiency observed in acrodermatitis enteropathica in humans (Moynihan, 1975). Furthermore, changes in immune reactivity (Fraker et al., 1979; Fernfindez et al., 1977; Chandra and Au, 1980; Fraker et al., 1982; Frost et al., 1981), as well as depressed levels of circulating thymic hormone (Iwata et al., 1979), have also been observed in experimental Zn depletion.

Zn induces per se mitogenic effects on human peripheral blood lymphocytes (Kirchner and ROhl, 1970; Berger and Skinner, 1979) which are monocyte-dependent (Rahl and Kirchner, 1978), and on murine spleen cells (Warner and Lawrence, 1986a). The metal is also able to modulate the immune response, up-regulating the proliferative response to T cell mitogens of spleen cells from low responder C57BL/6 strain mice (Malav6 and Rond6n, 1984). Supplementation of cultures with the metal also increases the in vitro antibody response of mouse spleen cells to both T-dependent and T-independent antigens (Malav6 et al., 1983). Furthermore, Zn promotes the differentiation and activation of human B cells and has synergistic effects with polyclonal activa- tors of human B lymphocytes (Cunningham Rundles et al., 1980). It has also been shown that zinc restores the plaque-forming cell response to sheep red blood cells (SRBC) in cultures of spleen cells from aged mice (Winchurch et al., 1984), an effect determined by the enhancement in the synthesis of IL-1 and IL-4 mediated by the metal (Winchurch et al., 1987).

The present study examines the capacity of Zn when added in vitro to modulate the proliferation of human lymphocytes in response to concanavalin A (Con A) and in the autologous mixed lymphocyte reaction (AMLR). In cultures of human peripheral blood lymphocytes stimulated with ZnC12 alone, we were also able to demon- strate that the metal was capable of inducing per se the appearance of interleukin-2 receptors (IL-2R) and transferrin receptors (TrR) at the cell surface, and that incubation with the monoclonal

antibody (mAb) anti-Tac reacting against the IL-2R inhibited the incorporation of [3H]- thymidine by the cultured cells. However, addition of exogenous IL-2 did not increase [3H]- thymidine incorporation in cultures supplemented with ZnC12.

Material and Methods

Mitogen stimulated cultures Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood obtained from normal volunteers. Following density-gradient centrifugation on Ficoll-Hypaque (density 1.077) for 30min at 400 × g, cells remaining at the interphase were collected, washed twice and resuspended in medium RPMI 1640 supplemented with antibiotics, 10~o fetal bovine serum (FBS) and 2 mM L-glutamine (all from Grand Island Biological Lab., Grand Island, NY) (complete medium). Thereafter, cells were counted and adjusted at a concentration of 2 × 10 6 per ml.

To study the effect of Zn on the proliferative response to Con A, 100-#1 aliquots of the PBMC suspension containing 2 × 105 cells were placed into the wells of fiat-bottomed 96-well micro- culture trays (Linbro, 76-002-05, Flow Labora- tories, Inc., McLean, VA, USA). Grade IV Con A (Sigma Chemical Co., St. Louis, MO) was dissolved in culture medium RPMI 1640, and 100/~1 of a suitable dilution were added to appro- priate wells to give a final concentration of either 10/~g or 20/~g of the mitogen per ml of culture. Parallel sets of cultures were added with 20 #1 of ZnC12 (Sigma Chemical Co., St. Louis, MO) at a final concentration of 1 × 10-4 M or with similar volume of physiological saline solution. Cultures were set up by triplicate and incubated for 72 h at 37 °C in a humid atmosphere of 5 ~o CO2 in air. To assess DNA synthesis, 0.5/~Ci of [3H]- thymidine (specific activity 2 Ci/mM) was added to each well 8 h before the end of the incubation period. Thereafter, cultures were collected by suction on glass-fiber filters, using a multiple

automated sample harvester. Radioactivity was measured in a liquid scintillation spectrometer and the results were expressed as the arithmetic means + SD of triplicate cultures.

When cultures were stimulated with Zn alone, 20/~1 of ZnC12 solution at a dilution suitable to give a final concentration in culture of 1 x 1 0 - a M ZnC12 were added to each well containing 2 x 105 cells in a volume of 200 #1. Cultures were incubated for different times up to 168 h, since preliminary experiments indicated that maximal DNA synthesis in response to Zn occurred between 120 and 168 h of culture. The incorporation of [3H]thymidine was assessed as described above.

Autologous mixed lymphocyte cultures Responder T and stimulator non-T lymphocytes were isolated from PBMC obtained from heparinized venous blood of normal donors as described above. Briefly, PBMC were mixed with SRBC pretreated with 2-aminoethylisothioro- nium bromide hydrobromide (Sigma Chemical Co., St. Louis, MO). After centrifugation at 1500 rpm for 10 min, the lymphocyte-SRBC pellet was gently resuspended, layered on Ficoll- Hypaque density 1.077, and the gradient was cen- trifuged for 30 min at 400 x g. Then, the rosetted T cells in the pellet were treated with 0.83~o ammonium chloride/0.17 M Tris buffer (pH 7.2) to lyse SRBC, and extensively washed. Non-T lymphocytes collected at the interphase were washed and suspended in complete medium. Purification ofresponder T lymphocytes was over 95 ~o as assessed by indirect inmunofluorescence with the mAb OKT3 (Ortho Diagnostic Systems, Raritan, NJ) and by detecting the number of cells beating surface Ig by direct immunofluorescence with a polyvalent goat antihuman immuno- globulin antiserum conjugated to fluorescein (Cappel Worthington Biochemicals, Malvern, PA).

Unidirectional AML cultures were established by triplicate in 96-weU round-bottomed micro- culture plates (Linbro 76-013-05, Flow Labora- tories, Inc. McLean, VA). Aliquots containing

1 x 105 T lymphocytes used as responder cells and 1 x 105 irradiated stimulator non-T lymphocytes (which have received 2000 rads in 10 min from a cobalt source) were distributed into each well. T responder lymphocytes and non-T stimulator cells were also separately cultured in triplicate as controls. Cultures were incubated for 6 days at 37°C in 5~o CO2 in a 100~o humidity environment, and the synthesis of DNA was assessed as described for mitogen-stimulated cultures. Parallel sets of cultures were supplemented with 20 #1 of a ZnC12 solution prepared to obtain a final concentration of ZnC12 in the culture of 10 - 4 M , o r with a similar volume of physiological saline solution. Results were expressed in Acpm, as the cpm obtained in control cultures of T responder lymphocytes incubated alone were subtracted from the cpm obtained in mixed cultures of T-responder and non-T stimulator cells.

Detection of receptors for IL-2 and transferrin Expression of the IL-2R was studied by immunofluorescence with the mAb anti-Tac kindly donated by Dr. Thomas A. Waldmann, NIH, Bethesda MD. The characteristics and specificity of this antibody have been described by Uchiyama et al., (1981). Expression of the TrR was assessed by immunofluorescence with the mAb OKT9 (Ortho Diagnostic Systems, Raritan, NJ). Briefly, cells collected on different days of culture were washed and adjusted to a concentration of 1 x 10 7 cells per ml in phosphate- buffered saline solution (PBS), pH 7.2, supplemented with 2~o FBS and 0.01~o sodium azide. Five #1 of the corresponding mAb were added to 100 #1 of the cell suspension and the mixture was incubated at 4°C for 30 min. Then the cells were washed twice with the PBS solution supplemented as specified above and mixed with 100 #1 of a goat antiserum to mouse immunoglobulins labeled with fluorescein (Ortho Diagnostic Sys- tems, Raritan, NJ). After 30 min incubation at 4 ° C, cells were washed twice and then examined in a Zeiss microscope equipped for epifluo- rescence. A minimum of 200 lymphoid cells per slide were counted by two independent observers.

Results

Effect of the addition of Zn on the proliferative response to Con A and on the A M L R

Preliminary experiments performed to see wheth- er the addition of ZnC12 in vitro was able to modify the proliferative response to Con A of normal human lymphocytes as well as the A M L R indicated that supplementation of cultures with either 5 x 10 -5 M or 1 x 10 - 4 M ZnC12 enhanced the uptake of [3H]thymidine in either system. The maximal potentiating effect was observed in cultures supplemented with ZnC12 at a final concentration of 10 4 M. As shown in Fig. 1, addition of 1 0 - 4 M ZnCI2 significantly enhanced the proliferative response of normal donor lymphocytes stimulated with either 10 or 20 #g of Con A per ml of culture in 13 and 12 consecutive experiments performed, respectively. The percent increase of the response showed an average + SE of 49 + 20% in cultures stimulated

E 105 n {.)

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0 Q _

Io 4

c

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p < 0.005 p < 0.005

/

I O f f g / m l 2 0 f f g / m l

Con A dose

Fig. 1. Proliferative response to Con A of normal human lymphocytes cultured in the absence (©) or in the presence ( Q ) of 10-4 M ZnC12. Bars represent the means + SE of the results obtained in 13 (10 #g/ml of Con A) and 12 (20 #g/ml of Con A) consecutive experiments. Statistical analysis of the results was performed by means of Student's t test for paired

data.

with 10 #g/ml of Con A and 59 + 19% in cultures stimulated with 20 #g/ml of Con A.

The concentration of Zn used here did not affect cell viability in cultures stimulated with Con A. Thus, the percentage of viable cells was similar in cultures incubated in the absence or in the presence of 1 × 1 0 - 4 M Z n C 1 2 (results not shown).

As shown in Fig. 2, supplementation of the culture system with 1 0 - 4 M ZnC12 markedly potentiated proliferation in the AMLR. The enhancing effect of Zn on the lymphoproliferative response to autologous 'self was observed in 14 consecutive experiments performed. The percent increase of the response showed an average + SE = 445 + 127 %. Interestingly, the potentiating effect of Zn on the incorporation of [3H]- thymidine in the AMLR was significantly more pronounced than Zn-mediated enhancement of the proliferative response to Con A.

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Fig. 2. Effect of the addition of ZnCI 2 on the proliferative response in the autologous mixed lymphocyte reaction: 105 responder T lymphocytes were cultured with 105 irradiated stimulator autologous non-T cells either in the absence ( O ) or in the presence ( O ) of 10-4 M ZnC12. Bars represent the means + SE of the results obtained in 14 consecutive experi-

ments. Statistical analysis as in Fig. 1.

Zn induces the appearance of 1L-2R and TrR on human lymphoid cells

As shown in Fig. 3, stimulation of human PBMC with 10 - 4 M ZnC12 alone induced a progressive increase in the percentage of cells expressing IL-2R as detected by indirect immunofluorescence with the mAb anti-Tac. The percentage of IL-2R + lymphocytes reached maximal levels on day 6 of culture, in parallel with metal-induced increase in [3H ]thymidine incorporation. Despite the decrease of the radioactive uptake observed at day 7 of culture, the percentage of lymphocytes beating IL-2R did not fall at this time and remained at levels similar to those on day 6 of culture, when, as stated above, maximal DNA synthesis was observed (Fig. 3).

Similar results were observed when the expression of TrR was analysed by indirect immunofluorescence with the mAb OKT9. Thus, a progressive increase in the percentage of cells expressing TrR in parallel with the increase in the incorporation of [3H]thymidine was observed in

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Fig. 3. Expression of the IL-2 receptor in cultures of normal human PBMC stimulated with ZnC12 as assessed by indirect immunofluorescence with the mAb anti-Tac. Hatched bars represent the percentage of IL-2R + cells in cultures incubated with 10 -4 M ZnC12 and open bars represent the percentage of IL-2R+ cells in control cultures incubated without ZnC12. Incorporation of [3H]thymidine (mean cpm + SE) in cultures stimulated with 10-aM ZnC12 ( ) and in non-stimulated control cultures

( . . . . . . ).

cultures stimulated with 10 - 4 M ZnCI 2. The percentage of cells bearing the TrR did not diminish as rapidly as [3H]thymidine incorporation, remaining high even on day 7 of culture (Fig. 4).

A discrete augmentation in the percentage of cells bearing receptors for IL-2 and transferrin also occurred in control cultures without ZnCI 2. However, in this case the increment in the percentage of cells showing those activation markers was significantly smaller than that observed in cultures stimulated with Zn (Figs. 3 and 4). Cells expresing IL-2R and TrR in non-stimulated cultures may represent T lymphocytes responding to autologous determinants on the surface of non-T cells in cultures of unfractionated PBMC.

Effect of the monoclonal antibody anti-Tac on the proliferative response to Zn

Since the appearance of an activation marker such as IL-2R at the cell surface does not defini- tively prove that the DNA synthetic response to

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Fig. 4. Expression of the transferrin receptor in cultures of normal human PBMC stimulated with ZNCL 2 as assessed by indirect immunofluorescence with the mAb OKT9. Hatched bars represent the percentage of OKT9 + cells in cultures incubated with ZnC12 10-4 M and open bars represent the percentage of OKT9 + cells in control cultures without ZnC12. Incorporation of [3H]thymidine (mean cpm+ SE) in cultures stimulated with ZnC1 z 10-aM (- ) and in non-stimulated control cultures

( ).

Zn requires binding of IL-2 to its receptor, occurring via an IL-2-dependent pathway, further experiments were designed to study the effect of the blockade of the IL-2R on the proliferative response to the metal. Incubation of cells with the mAb anti-Tac, at a final 1 : 500 dilution in the culture, markedly inhibited the incorporation of [ 3H ]thymidine by human lymphocytes stimulated with 10 - 4 M ZnC12. Inhibition of the proliferative response induced by Zn reached 72% and 74% in the two experiments presented (Fig. 5).

Effect of the addition of human rlL-2 on Zn-induced lymphocyte proliferation

Further experiments were designed to measure the effect of the addition of rlL-2 on lymphocyte proliferation induced by zinc. As shown in Table I, when the uptake of [3H]thymidine was evaluated at either day 5 or 6 of culture, the addi-

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Fig. 5. Inhibition of the incorporation of [3H]thymidine in cultures of normal human PBMC stimulated with 10 4 M ZnC12 and treated with the mAb anti-Tac. Results are expressed as mean cpm + SE of triplicate cultures. Open bars represent the incorporation of [3H]thymidine in cultures incubated without anti-Tac and hatched bars represent the incorporation of [3H]thymidine in cultures incubated with

anti-Tac at a 500-fold final dilution.

tion of 125 U/ml of culture of human rIL-2 (Human Interleukin-2, Recombinant, Sigma Chemical Co., St. Louis, MO) increased the spontaneous incorporation of [3H]thymidine in cultures without ZnC12, in most of the cases. However, in cultures stimulated with either 5 x i 0 - 5 M or 1 x 10- 4 M ZnC12, the addition of rIL-2 did not produce marked variations in the uptake of [3H]thymidine. Thus, the stimulation index (S.I.) was depressed in cultures with rIL-2 as compared with that observed in cultures performed in the absence of exogenous IL-2 (Table I). Similar results were obtained when cultures were supplemented with 250 U/ml of rlL-2 (results not shown).

Discussion

The capacity of Zn to enhance Con A-induced proliferation of human PBMC observed here is in agreement with previous work demonstrating that addition of Zn in vitro potentiates the response to T cell mitogens of spleen cells from C57BL/6 mice (Malav6 and Rond6n, 1984), as well as with the enhancing effect of Zn-transferrin on the proliferative response to phytohemagglutinin of normal human lymphocytes (Phillips and Azari, 1974). An increase in the mitogenic response of lymphocytes from humans (Duchateau et al., 1981) and experimental animals (Gaworski and Sharma, 1978) has also been observed upon supplementation with zinc in vivo.

Zn is a cofactor of the active form of serum thymic factor (Dardenne etal., 1982), and increased numbers of immature lymphocytes have been found in the spleen of Zn-deficient mice (Nash et al., 1979). In view of the above, it may be thought that addition of ZnCI z to the cultures could promote the maturation of the less differen- tiated lymphocytes, rendering more cells responsive to the mitogen, therefore increasing the response to Con A. The direct mitogenic activity of Zn (Kirchner and Rtihl, 1970; Hart, 1978) could also have additive effects with Con A-induced proliferation. In addition, since macro-

7

TABLE I

Effect of the addition of human rlL-2 on the incorporation of [3H]thymidine in cultures of normal human PBMC stimulated

with ZnC12

Day of culture Final concentrat ion of added Z n C 1 / ( M ) [3H]Thymidine incorporation

Without IL-2 With IL-2 a

Mean cmp + SE S.I. Mean cmp + SE S.I.

Expt. 1 5

Expt. 2 5

Expt. 3

5

- 602 + 162 2165 + 181

5 × 10 -5 7351 + 1460 10.2 7074 + 309 3.3

1 x 10 -4 7610 + 315 11.6 7700 + 718 3.6

- 744 + 249 3835 + 879

5 x 10 -5 11981 + 943 16.1 10996 + 240 2.9

1 × 10 -4 10476 + 681 14.1 12536 + 1698 3.3

- 3 5 5 + 26 5 0 2 + 51

5 x 10 -5 5223 + 1179 14.7 4871 + 744 9.7

1 x 10 - 4 2398 + 356 6.7 2552 + 111 4.1

- 248 + 31 240 + 35

5 × 10 -5 4885 + 704 19.7 2075 + 443 8.7

1 × 10 - 4 1030 + 214 4.2 1056 + 202 4.4

- 2880 + 171 4408 + 377

5 x 10 -5 11518 + 1049 4.0 13969 + 1193 3.2

1 x 10 -4 18540 + 1304 6.4 16113 + 1160 3.7

- 4729 + 943 5624 + 322 5 × 10 -5 16068 + 2429 3.4 15142 + 2069 2.7

1 x 10 - 4 21657 + 4237 4.6 17797 + 714 3.2

" H u m a n rlL-2 was added at a final concentrat ion of 125 U per ml of culture.

phages depress lymphocyte proliferation in cultures stimulated by relatively high doses of mitogen, such as those used here (Baird and Kaplan, 1977; Malavr, 1981), the capacity of Zn added in vitro to inhibit the function of activated macro- phages (Chvapil et al., 1977) could contribute to increase the response to Con A in cultures supplemented with the metal.

Addition of Zn in vitro produced a striking enhancement of the AMLR, which could result from the cumulative response to the metal and to autologous non-T cells, since both induce maximal synthesis of DNA on day 6 of culture (Rtlhl et al., 1971). The enhancing effect of the metal on the AMLR could also be determined by its capacity to stimulate the synthesis of IFN- 7

(Salas and Kirchner, 1987), which is a potent inducer of Ia expression.

Alteration of self constituents by heavy metals leading to immune reactivity against the modified self has been postulated (Pelletier et al., 1985). Thus, the important potentiating effect of Zn on the AMLR observed here could be related to its potential capacity to modify the conformation of self Ia (Warner and Lawrence, 1986b). In any case, the striking modulatory effect of Zn on the AMLR observed here indicates that the metal participates in physiological immunoregulation.

Zn potentiates the response to PHA and Con A of spleen cells from C57BL/6 mice, which are low responders to T cell mitogens, but only slightly modifies the response of spleen cells from

high responder BALB/c mice (Malav6 and Ronddn, 1984). Similarly, Zn enhanced low plaque-forming cell responses generated in cultures without 2-mercaptoethanol but not high antibody responses generated in cultures with 2-mercaptoethanol (Malav6 et al., 1984). It has also been observed that supplementation with Zn in vivo enhanced mitogenesis in individuals showing low proliferative responses before the administration of the metal (Duchateau et al., 1981). It appears therefore that Zn exerts immunopotentiating effects when the magnitude of the basal response of the system is low. Thus, relatively low proliferative responses such as the AMLR could be more susceptible to the potentiating effects of Zn than the strong proliferative response to a polyclonal activator of T cells such as Con A.

Alternatively, the enhancing effect of Zn on lymphocyte proliferative responses could be related to the capacity of this metal to increase the synthesis of IL-4 (Winchurch et al., 1987), which could in turn activate the proliferation of B lymphocytes recruiting more cells to synthesize DNA.

The progressive appearance of cells bearing activation markers such as IL-2R and TrR in cultures of human lymphocytes stimulated with ZnC12 alone observed here indicates that Zn induces progression in the cell cycle and that the increase in the incorporation of [3H ]thymidine in metal-stimulated cultures does not mainly result from the induction ofgene amplification or DNA repair. These observations are in agreement with previous work demonstrating that Zn determines blast transformation of mouse lymphocytes as assessed by staining with acridine orange (Warner and Lawrence, 1986a).

It has been shown that IL-2 produced upon T cell activation up-regulates the expression of IL-2R bearing anti-Tac reactive epitopes (revised by Smith, 1988). Thus, the progressive appearance of IL-2R on the surface of cells cultured in the presence of Zn not only indicates cell activation and progression in the cell cycle, but also suggests production of IL-2 within the culture system. The TrR appears later than the IL-2R,

requiring the presence of both functionally active IL-2R and IL-2 (Neckers and Cossman, 1983). So, the increase in the percentage of lymphocytes bearing TrR in cultures stimulated with ZnCI 2 alone indicates further progression in the cell cycle as well as production of IL-2 by the cultured cells.

Even though IL-2 plays a necessary role in lymphocyte proliferation in response to either specific antigens or mitogens (revised by Smith, 1988), a significantly less common IL-2-independent pathway of lymphocyte activation has also been observed (Koretzki et al., 1983; Laing and Weiss, 1988).

The mAb anti-Tac specifically blocks the binding of IL-2 to its receptor, inhibiting IL-2- dependent proliferation of human T cells (Miyawaki etal., 1982; Depper etal., 1984). Thus, the inhibition of the proliferative response to Zn by the mAb antiTac observed here might indicate that the mitogenic effect of the metal requires the binding of IL-2 to its receptor and suggests that it mainly occurs via an IL-2- dependent pathway.

A decreased S.I. was observed here in cultures stimulated with ZnC12 and containing exogenous IL-2. Such a finding could be explained by the marked augmentation in the uptake of [3H]- thymidine in control cultures without ZnC12 but supplemented with rIL-2. In fact, IL-2 alone is able to induce the expression of IL-2R as well as the proliferation of thymocytes and T cells which have not been exposed to mitogens or other inducer agents (Reem et al., 1985). On the other hand, exogenous IL-2 was unable to increase the proliferative response of cultures stimulated with ZnCI 2. Previous authors postulated that metal- induced mitogenesis ofmurine splenocytes resem- bles the AMLR, since both show similar kinetics, require CD4 + cells and are abolished by treat- ment with anti-Ia antibodies plus complement (Warner and Lawrence, 1986b). Lymphocyte proliferation in cultures of human PBMC stimulated with Zn could result from an amplification of the response of CD4 + cells to autologous antigens alone or in combination with the

response to metal-modified self antigens. It has recently been shown that T4 + cells become refractory to the growth-promoting effect of IL-2 before the disappearance of adequate levels of IL-2R (Gullberg and Smith, 1986). Furthermore, autoreactive lymphocytes which proliferate in the AMLR resemble 'naturally' activated T cells, which have also been shown to become refractory to IL-2 in vitro (Bandeira et al., 1987). IL-2 refrac- toriness of responsive cells could also explain the absence of an enhancing effect of exogenous IL-2 on the proliferative response to Zn, even though this requires binding of IL-2 to its receptor.

Acknowledgements

The authors express their thanks to Leslie Baute for his excellent technical help and to Lubomira Rybak for her expert secretarial work.

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