- Email: [email protected]
Stimulation of murine lymphocyte responses by cations
CELLULAR
101,425-439 (1986)
IMMUNOLOGY
Stimulation
of Murine Lymphocyte
GARVIN L. WARNERANDDAVID
Responses
by Cations’
A. LAWRENCE
Department of Microbiology and Immunology, Neil Hellman Medical Research Building, Albany Medical College of Union University, Albany, New York 12208 Received February 13, 1986; accepted May 5, 1986 The capacity of the heavy cations Pb, Ni, and Zn to modulate murine in vitro lymphocyte responses was examined. Pb and Ni ( 100 @4) were shown to enhance the in vitro plaque-forming cell (PFC) response to sheep red blood cells while 100 &f Zn had inhibitory effects.Each metal was able to stimulate the proliferation of murine splenocytes as determined by [‘H]thymidine incorporation and autoradiography. The enhancing effect of the metals on the PFC response was observed whether the results were expressed on a per culture or a per cell basis, indicating an actual increase.in B-cell differentiation. Both the PFC response and the proliferative response were shown to be sensitive to the type of medium employed (M- 199 gave optimum results) and to the presence or absence of 2-mercaptoethanol. As in autologous mixed-lymphocyte responses peak proliferation occurred after Day 5 in culture, was cell density dependent, and required the presence of both T cells and Ia+ cells. Treatment of splenocytes with anti-Thy-l .2, anti-Lyt- I, or anti-L3T4 plus complement completely abrogated the proliferative response, indicating that a Lyt-l+, Lyt-2-, L3T4+ T-cell was required for the induction of proliferation. The data are consistent with the hypothesis that the metals are capable of modifying the immune response directed at self either by directly altering self constituents (class II) or by modulating the autologous T-cell response. 0 1986 Academic press, Inc.
INTRODUCTION The heavy cations Pb, Ni, and Zn have been shown to modulate immune responses and in vitro (4-6). Although Zn and Ni (in rats) are essential trace elements (7) and Zn deficiency causes severe immune abnormalities (8), these metals have been suggested to be carcinogens (9, 10). Furthermore, they have been shown to elicit contact dermititis reactions (11) presumably by interacting with and altering self constituents and rendering them immunogenic. As such, the association of these metals with host constituents has important health implications. The mechanism whereby these metals exert their immunomodulatory effects is not known. Ni and Zn stimulate DNA synthesis of human peripheral blood lymphocytes ( 12- 14). Ni also is known to stimulate DNA synthesis of human thymocytes ( 15). The degree of Ni stimulation has been shown to correlate ( 16) with the Ni sensitivity of individuals although some overlap between control and N&sensitive patients has been observed ( 17). In addition to the mitogenic activities ofNi and Zn, these metals have been shown to restore the proliferative response of lymphocytes exposed to the metal chelain vivo (l-3)
’ This work was supported by NIH Grant ES 03 179.
425 0008-8749/86$3.00 copyright 8 1986 by Academic I’res, Inc. AU rights of reproduction in any fom reserved.
426
WARNER
AND
LAWRENCE
tors EDTA (18) and phenanthroline ( 19). Pb, Ni, and Zn induce murine splenocyte DNA synthesis (5,6,20). Pb and Ni have been demonstrated to enhance DNA synthesis induced by the B-cell mitogen LPS* (5,6), while Pb has been reported to enhance Con A- and PHA-induced T-cell proliferation (2 1). In terms of functional immunity, Pb and Ni have been shown to enhance in vitro humoral immune responses, while Zn was inhibitory (5). Furthermore, the enhancement of the in vitro PFC response by Pb occurred in the absence of T cells (6). It appears that these metals have pleiotropic effects and are capable of modulating both T-cell and B-cell activities. The ability to discriminate between self and nonself is central to the role of the immune system. As such, the immune system is delicately balanced between positive and negative regulatory influences. Agents which have the capacity to upset this delicate balance may affect the health of the host by decreasing resistance to pathologic agents or by contributing to the generation of autoimmune disease. In a recent report by Pelletier et al. (22) it was demonstrated that HgCl, treatment of the Brown-Norway rat altered resident lymphocytes in such a manner that they were able to stimulate in vivo lymphocyte proliferation when transferred to an autologous host, the hypothesis being that the metals interact with and alter self constituents, thereby rendering these self antigens immunogenic. Ni-specific T-cell clones were recently isolated from peripheral blood of Ni contact-sensitive patients (23); these T-cell clones responded to Ni in an MHC class II-restricted fashion. Hg is quite toxic in the mouse and displays none of the immunomodulatory activities ascribed to Pb, Ni, and Zn (5); however, the effect of Hg in the rat and Ni in the human may serve as models whereby some of the immunomodulatory activities of Pb, Ni, and Zn in the mouse may be explained. In this investigation, the in vitro effects of Pb, Ni, and Zn on murine splenocyte proliferation have been further defined and examined. Different tissue culture media were compared for their ability to sustain metal-induced modulation of both in vitro PFC response and proliferation, and the requirement for 2-mercaptoethanol(2ME) was determined. The kinetics of lymphoproliferation was measured for extended culture periods. Peak responses were observed late (Days 5-7). Cellular contact was also implicated as being important in the overall response mediated by these metals. Lymphocyte subset depletion experiments were performed to determine whether T cells, B cells, and/or IaC cells were required for these metals to induce proliferation. The results presented herein are consistent with the hypothesis that Pb, Ni, and Zn modulate the in vitro murine immune response by altering the immunogenicity of self, possibly by modifying class II MHC molecules. MATERIALS
AND METHODS
Animals. Female CBA/J mice (Jackson Laboratory, Bar Harbor, Maine) 2-4 months of age were used for all studies. Mice were maintained on laboratory chow and acidified, chlorinated water, pH 3.0, ad libitum. Media and reagents.RPMI-1640, M- 199 (with Hanks BSS), and MEM were purchased from M. A. Bioproducts (Walkersville, Md.) and were supplemented with 5% * Abbreviations used: 2ME, 2-mercaptoethanol; PBS, phosphate-buffered saline; BSS, balanced salt solution; Con A, concanvalin A, LPS, lipopolysaccharide; MHC, major histocompatability complex; PFC, plaque-forming cell; SRBC, sheep red blood cell; FBS, fetal bovine serum; GSH, glutathione; AMLR, autologous mixed-lymphocyte response; IFN, interferon; IG 1, interleukin 1; TRF, T-cell replacing factor.
CATIONIC
IMMUNOMODULATION
427
FBS (co.8 rig/ml endotoxin; Sterile Systems, Logan, Utah), L-glutamine (1 mM), Napyruvate (1 mM), nonessential amino acids (0.1 m&Q, gentamicin (50 pg/ml), and NaHC03. 2ME (5 X 10e5 A& Eastman Kodak, Rochester, N.Y.) was included in all media unless otherwise indicated. Stock solutions of PbCl*, NiCl*, and ZnClz (Fisher Scientific, Rochester, N.Y.) were prepared in physiologic saline at a concentration of 1 x 10m2 M. Sheep red blood cells (SRBC) were purchased from Colorado Serum (Boulder, Colo.). Monoclonal antibodies. The following hybridoma cell lines were obtained from the American Type Culture Collection (Rockville, Md.): 30-H 12 (anti-Thy- 1.2, rat IgG), 3.115 (anti-Lyt-2, rat IgM), GK1.5 (anti-L3T4a, rat IgG), and HO-13-4 (anti-Thy1.2, mouse IgM). Cells were cultured in the recommended media, and the supernatants harvested 7 days following entry into stationary phase and dialyzed into phosphate-buffered saline (PBS, pH 7.4). Each preparation was titered against thymocytes using rabbit complement and found to have a 50% cytotoxic titer greater than 1: 1000. For depletion experiments the rat monoclonal reagents were used at a final dilution of 1: 10. The supematant from HO-l 3-4 was precipitated with 50% ammonium sulphate and reconstituted to 10% of its original volume with PBS. Primary in vitro PFC assay, Spleens were aseptically removed and a single cell suspension was made by pressing the spleens between the frosted ends of two microscope slides. Clumps were allowed to settle and cells were cultured as described (24). Briefly, 5 X lo6 spleen cells/O.5 ml, plus or minus antigen (SRBC), plus or minus metal, were cultured for 5 days in special gas composed of 10% C02, 7% 02, 83% N2. On Day 5, cells of triplicate cultures were harvested and pooled and the number of plaque-forming cells (PFC) was determined by use of a modification of the Jeme plaque assay (25). Only direct plaques were detected. Proliferative assay. Metal stocks were appropriately diluted with saline and 25 ~1 was added to the appropriate wells of 96-well flat-bottom tissue or round-bottom culture plates (catalogue No. 3596 or 3799, respectively; Costar, Cambridge, Mass.), such that the final concentration of metal was between 5 and 100 PM, as indicated in the text. Zero metal controls received saline alone. A single cell spleen cell suspension (0.2 ml) was added such that each well contained 4 X lo4 to 8 X lo5 cells/well. Cells were cultured in special gas composed of 10% C02, 7% 02, 83% N2. On the day of harvest 0.5 &i of [3H]thymidine (New England Nuclear Corp.), 25 ~1, was added and individual wells were harvested 6 hr later using a multiple cell culture harvester (Skatron AS, Liebgen, Norway). Filter disks were dried and counted in a Nuclear Chicago Isocap 300. Lymphocyte subpopulation separations. T cells were isolated by panning on antimouse immunoglobulin-coated plates as previously described (26). Briefly, viable splenocytes were isolated on a Ficoll-metrizoate gradient (density 1.090) (27) and suspended in PBS containing 10% heat-inactivated FBS and 0.1% NaN3 at a concentration of 10’ cells/ml; 3 ml of this suspension was placed on an 18-cm bacteriologic petri dish that previously had been coated with rabbit F(ab’)2 anti-mouse immunoglobulin. The unbound cells were gently collected and the plates gently washed once with PBS to maximize recovery. The cells were then washed 3~ with BSS (balanced salt solution, pH 7.4) containing 2% FBS. B cells were obtained from animals that had been injected with 50 ~1 anti-mouse thymocyte serum (M.A. Bioproducts) 48 hr prior to exsanguination. A single cell suspension of splenocytes was obtained, resuspended at l-4 X 10’ cells/ml in BSS,
428
WARNER
AND LAWRENCE
2.5 9 $ x
2
2
1.5
2 3 2
1
y
0.5
5#n8
Pb
*
B
1. Modulation of the in vitro PFC response by Pb, Ni, and Zn: comparison of MEM (dotted), M- 199 (slashed), and RPMI-1640 (cross-hatched). Splenocytes (5 X lo6 cells/well) were cultured in the presence or absence of 1 X 1Oe4M Pb, Ni, or Zn as indicated. On Day 5 cells were harvested and assayed for PFCs as described under Materials and Methods. The data presented are. representative of three separate experiments. FIG.
containing 2% FBS, and subjected to two cycles of anti-Thy- 1.2 ( 1:25; HO- 13-4) and anti-Lyt- 1.1 ( 1: 125; CE/J anti-CBA/J alloantiserum; 30 min, 4”C), plus rabbit complement (1:25; 37°C 45 min). The cells were spun through 100% FBS and washed 3X with BSS containing 2% FBS. All other antibody plus complement treatments were performed using a one-cycle protocol as follows. Antibodies were used at a final dilution of 1: 10 except for antiIak (A.TH anti-A.TL alloantiserum) and anti-Lyt- 1.1 (CE/J anti-CBA/J alloantisera) which was used at a fin&lilution of 1:25. Cells were incubated with antibody for 30 min, 4°C pelleted, and resuspended in rabbit complement (1:25) for 45 min, 37°C. The cells were then centrifuged through 100% FBS and washed 3X with BSS containing 2% FBS. Autorudiogruphy. Cells were cultured and pulsed as in the proliferative assay. Cells were harvested into 1.5-ml polypropylene microcentrifuge tubes, centrifuged through 100% FBS, washed 2X with PBS, and cytocentrifuged onto clean glass slides. The slides were then air dried, fixed with 1% gluteraldehyde, and emulsed using NTB-2 (Eastman Kodak). The slides were exposed for 1 week at 4°C developed using developer D-19, fixed with a 25% solution of Na-thiosulfate and stained with Wright’s stain. Labeled nuclei were counted by light microscopy under oil. Statistical analysis. Results are expressed as means + SD. The difference between means was assessed by the two-tailed Student t test. Differences were considered significant when P was greater than 0.05. RESULTS Characterization
of the in Vitro Primary
Response
The ability of RPMI-1640, M-199, and MEM to sustain an in vitro primary immune response was determined. Each basal medium was supplemented equivalently as described under Materials and Methods. As demonstrated in Fig. 1, the enhancing effects of Ni and Pb were observed regardless of the medium employed; however, the degree of metal enhancement was significantly greater in M- 199 compared with either RPMI-1640 or MEM. The inhibition exhibited by Zn was apparent in each medium as well. M-199 appears to be the medium of choice when observing metal-
CATIONIC
429
IMMUNOMODULATION TABLE 1
Effect of Metals on Cell Recovery and the in Vitro PFC Response Metal 1x 5x 1X 5X 1X 5X
0 10-4MPb lO-5 MPb 10e4MNi IO-‘MNi 10m4MZn 10e5MZn
Cell recovery’l (X106) 6.42 7.51 5.50 8.40 6.61 10.70 6.90
PFC/cuIture 640+23 2080 + 224* 1756 f 289* 1320 f 100* 716k33 289 f 73* 577 + 76
(l.OO)b (3.25) (2.74) (2.06) (1.12) (0.45) (0.90)
PFC/ lo6 cells 299.1 830.9 957.8 471.4 325.0 81.1 251.0
f 11 f 89* 2 158* f 36* f 15 f 20* + 33
(1.00) (2.78) (3.20) (1.58) (1.09) (0.27) (0.84)
Note. CBA/J splenocytes were cultured in M- 199 containing 5% FBS at a density of 5 X lo6 cells/O.5 ml. After 5 days in culture triplicate wells were pooled and the number of direct PFCs enumerated. a Cell counts were made on pooled triplicate wells and are expressed as total number of cells recovered per three wells. b Number in parentheses indicates stimulation index (metal treated/O metal control). * P < 0.05 relative to 0 metal control.
induced immunomodulation of the in vitro PFC response. It should be noted that there were no differences in either cellular viability or cell recovery among the various media (data not shown). Each of the metals tested caused an increase in the number of recoverable cells compared with the saline controls (Table 1). Even though Zn inhibited the PFC response, it enhanced cell recovery. The enhanced PFC response by Pb and Ni was observed regardless of whether the PFC results were expressed on a per culture or a per cell basis (Table l), indicating that the enhancement was due to an actual increase in the number of differentiated B cells rather than solely due to enhanced recovery. Pb increased the PFC response in the absence of SRBC; however, as previously reported (5), the magnitude of the enhancement of the specific response was always greater than the nonspecific response.
Characterization of Metal-Induced Lymphocyte Proliferation The ability of RPMI- 1640, M- 199, and MEM to support metal-induced lymphocyte proliferation was assessed. Spleen cells were stimulated with optimal metal concentrations and were pulsed and harvested on Day 6. As demonstrated in Fig. 2, Ni was capable of inducing significant [3H]thymidine incorporation regardless of the basal medium employed. Pb and Zn did not induce significant [3H]thymidine incorporation in MEM without 2ME (Fig. 2A). It should be noted that the absence of a response could not be accounted for by a shift in kinetics (data not shown). Regardless of the basal medium employed, the proliferative response induced by each of the metals was greater in the presence of 2ME (Fig. 2B). M-199 consistently supported metal-induced responses which were significantly greater than the response observed with MEM. M- 199 supported a significantly greater Zn-induced response compared with RPMI-1640 while the difference between M-199 and RPMI-1640 with respect to Pb and Ni stimulation was not significant; however, M- 199 did consistently produce a higher proliferative response regardless of the medium employed. In order to be sure that we were indeed looking at [3H]thymidine incorporation into nuclear
430
WARNER
AND LAWRENCE
FIG. 2. Metal induction of lymphocyte proliferation, comparison of MEM (dotted), M-199 (slashed), and RPMI- 1640 (cross-hatched). Splenocytes (5 X 10’ cells/well) were cultured in the presence or absence of 1 X 10e4 M Pb, Ni, or Zn as indicated. On Day 5 the wells were pulsed with 0.5 pCi of [3H]thymidine for 6 hr, harvested onto glass fiber mats, dried, and counted by liquid scintillation spectroscopy. Responses were assayed in the absence (A) or presence (B) of 5 X IO-’ A4 2ME. The data presented are representative of three separate experiments.
DNA, autoradiography of whole cells pulsed with [3H]thymidine was performed. As indicated in Table 2, Pb, Ni, and Zn each caused an increase in the percentage of labeled nuclei, and this increase corresponded to the relative degree of thymidine incorporation assessed by liquid scintillation counting. In addition, cell viability in the presence of metal was equal to or greater than the control group. Taken together these data demonstrate that the metals Pb, Ni, and Zn induce murine lymphocytes to initiate nuclear DNA synthesis late in culture. TABLE 2 Effect of Metals on Viability and [3H]Thymidine
Incorporation into Nuclear DNA
% Viable“
% Labeled nucleib
61 15 81 59 82 65 64
12 20 ND’ 33 ND 36 ND
Metal 1x 5x 1X 5X 1X 5X
0 10-4MPb lo-‘MPb 10m4MNi IO-‘MNi lo-‘+MZn lo-‘MZn
Note. CBA splenocytes were cultured in the presence or absence of metal at a density of 5 X 10’ cells/ well. On Day 6 individual wells were pulsed with 0.5 &i [‘H]thymidine for 6 hr and harvested into polypropylene tubes. ’ Viability was assessedby exclusion of trypan blue. b Cells were cytocentrifuged onto clean glass slides, fixed, stained, and emulsed with NTB-2. Slides were developed 7 days later and labeled nuclei counted by light microscopy. ’ ND indicates that value was not determined.
CATIONIC
431
IMMUNOMODULATION
120 n k
I#) m 60
2x 2s 9 8" 20 0
LPS
Con
A
3. Effect of metals on mitogen-induced lymphocyte proliferation. Splenocytes (2 X 10’ cells/well) were cultured in the presence of LPS (10 &ml) or Con A (2.5 &ml) as indicated. Cells were also stimulated with 0 (open), 1 X 10m4M Pb (slashed), 1 X 10m4M Ni (cross-hatched), or 1 X 10m4M Zn (closed). On Day 2 individual wells were pulsed with 0.5 &i of [‘Hlthymidine for 6 hr, harvested onto glass fiber mats, dried, and counted by liquid scintillation spectroscopy. The data presented are representative of three separate experiments. FIG.
Eflect of Heavy Metals on Con A- and LPS-Induced Mitogenesis In order to determine whether Pb, Ni, and Zn had a generalized capacity to modulate lymphocyte proliferation, we examined the ability of these metals to alter mitogen-induced proliferation. Figure 3 demonstrates that Pb, Ni, and Zn did not significantly enhance nor inhibit LPS- or Con A-induced proliferation.
Kinetics of Metal-Induced Proliferation Earlier studies (6) had indicated that Pb caused an early (Day 3) increase in [3H]thymidine incorporation. This early proliferative effect has been difficult to repeat consistently and probably was not due to Pb alone, in that it was dependent on the lot of FBS used (28). Pb enhancement of the PFC response has been consistent and is assayed for relatively late in culture (Day 5). Figure 4 shows the extended kinetics of metal-induced [3H]thymidine uptake by cultured mu&e splenocytes covering Days 2-9. Four doses of metal were employed in these studies, 1 X lop4 A4, 5 X 10F5 M, 1 X IO-’ M, and 5 X lop6 M, except in the case of Zn where previous work had demonstrated that concentrations of less than 1 X 1Oe5M had no effect. As shown in Figure 4A, Pb caused a dose-dependent proliferative response which peaked on Day 7 at approximately 90,000 cpm with a Pb dose of 1 X lop4 M. Ni (Fig. 4B) showed a dose-related response which peaked on Day 6 or 7 ( 1 X 10e4 It& 100,000 cpm). Zn (Fig. 4C) induction of the proliferative response occurred earliest (100,000 cpm on Day 6 with a dose of 1 X 10e4 M). The induction of proliferation by Zn always occurred earlier than that by Pb or Ni. Pb-induced proliferation sometimes peaked on Day 6 rather than Day 7 and usually was less than that of Ni and Zn in terms of the absolute number of cpm. The subtle variations in kinetics may have been due to minor variations in the final metal concentration or to minor variations in the final number of cells per well. Visual examination of the cultures revealed the appearance of small clusters of cells, approximately 3 days postinitiation of culture, the number of which appeared to correlate with intensity of the [3H]thymidine incorporation. The kinetics of the response are similar to other in vitro immune responses
432
WARNER
AND LAWRENCE
50
10 5
100 x
2 c3 3 P if!
50
10 100
50
10 2
3
4
5 6 OAYSlNCUlTURE
7
6
9
FIG. 4. Metal induction of lymphocyte proliferation: extended kinetics. Splenocytes (5 X lo5 cells/well) were cultured in complete M-199 containing 5 X lo-’ MZME in the presence of 0 (open squares), 1 X 10e4 M (closed squares), 5 X 1Oe5M (closed triangles), 1 X I Om5M (closed circles), or 5 X 10m6M (open circles) Pb (A), Ni (B), or Zn (C). On the indicated days the wells were pulsed with 0.5 pCi of [3H]thymidine for 6 hr, harvested onto glass fiber mats, dried, and counted by liquid scintillation spectroscopy. The data presented are representative of three separate experiments.
which require cell-cell contact such as the autologous or allogeneic mixed-lymphocyte responses.
Eflect of Cell Density on Metal-Induced Mitogenesis The influence of cell-cell interaction on the ability of these metals to induce lymphocyte proliferation was examined. Splenocytes placed in flat-bottom wells settle out randomly and are relatively evenly dispersed within the well. Round-bottom wells allow for the settling of the cells into the center of the well forming a relatively tight “button.” If cell-cell interactions are important in order to initiate the response, then as the cells become more dilute (sparse) in the flat-bottom wells a critical density is reached where the response drops off dramatically. In the round-bottom wells, the geometry of the well maximizes cell-cell contact; thus the response should fall off gradually, directly proportional to the number of cells per well. Figure 5 demonstrates the results of one such cell density experiment comparing round- and flat-bottom wells. Splenocytes were cultured at densities ranging from 8 X 1O5to 4 X 1O4cells per well. The response in flat-bottom wells dropped off markedly at approximately 1
CATIONIC
00
433
IMMUNOMODULATION
40
20 CELLS/WELL
10
S
4
x 1O‘4
5. Effect of cell density on cation-induced proliferation. Splenocytes were cultured in M-199 at densities ranging from 4 X lo4 to 8 X lo4 cells/well in either round-bottom [closed circles] or flat-bottom [closed squares] wells. Individual wells were stimulated with 100 pM Pb (A), Ni (B), or Zn (C). Cultures were pulsed with 0.5 #Ji of [3H]thymidine on Day 6 and harvested 6 hr later. The data presented are representative of three separate experiments. FIG.
X lo5 cells per well in the case of Ni and at approximately 2 X lo5 cells per well for both Pb and Zn. The decreased response in the round bottom wells is clearly more gradual. The peak [3H]thymidine incorporation was less in round-bottom wells than in flat-bottom wells; however, the kinetics of the response did not appear to change with regard to the type of plate used (data not shown).
Subpopulations
of Lymphocytes Requiredfor
the Proliferative
Response
The ability of Pb, Ni, and Zn to induce the proliferation of T cells, B cells, or Iacells was investigated. B cells were prepared by vigorous depletion of T cells, utilizing a combined treatment of in vivo rabbit anti-mouse thymocyte serum and two in vitro cycles of antibody plus rabbit complement as described under Materials and Methods. T cells were prepared by panning on anti-mouse Ig-coated petri plates as described by Wysocki and Sato (26). These preparations routinely contained fewer than 2% contaminating cells and often contained undetectable numbers of the eliminated population of cells as determined by flow cytofluorometry. As shown in Table 3, the B-cell preparations failed to proliferate in response to Pb, Ni, or Zn, whereas the T-cell preparations proliferated in response to the metals.
434
WARNER
AND LAWRBNCE TABLE 3
Metal Stimulation of Lymphocyte Subpopulations cpm/cultun? Cell preparations0
None
Pb
Unseparated T cells B cells T cells + B cells Anti-Ia-treated T cells Anti-Ia-treated splenocytes
8055 + 1404 705922519 2025 1 407 50582 126 169+ 43* 26lk 57”
35,392 + 66,682 k 918k 19,195 f 16Ok 206+
8725 6673* 128* 8824 19* 64*
Ni
Zn
7 1,932 +- 4960 84,466 + 5868* 1,390 + 137* 64,926 + 1194 1,068 + 720* 269+ 86;
48,459 + 6565 90,691 I!Z9238” 535* 133* 42,065 +- 4162 784+ 325* 134+ 85*
Note. Cells were cultured in flat-bottom 96-well plates (5 X IO5 cells/well), in M-199 containing 5% PBS, and were pulsed and harvested on Day 6. Cultures were stimulated with 100 &metal in saline. a T cells were obtained by depletion of B cells on anti-mouse Ig-coated plates. B cells were obtained from animals that were injected with ATS 48 hr prior to exsanguination and were subjected to two cycles of anti-Thy- 1.2 and anti-Lyt- 1.2 plus complement treatment. T cells and B cells were mixed at a ratio of 1: 1. Ia+ cells were depleted by one cycle of anti-Ia (A.TH anti-A.TL alloantisera) plus complement treatment. b The results represent the means of triplicate cultures + SD and are representative of three separate experiments. * P < 0.05 relative to respective unseparated control.
Clearly, T cells are required for the induction of proliferation by Pb, Ni, and Zn. The ability of anti-Ia treatment to abrogate the metal-induced proliferative response was also examined. As demonstrated in Table 3, treatment of either unselected splenocytes or T cells with anti-Ia plus complement completely eliminated the ability of these metals to induce proliferation. In order to further delineate the subpopulation of T cells required for the metals to stimulate lymphoproliferation, experiments employing anti-T-cell monoclonal antibodies were performed. As indicated in Table 4, anti-Thy- 1.2, anti-Lyt- 1.1, and antiTABLE 4 Effect of T-Cell-Specific Monoclonal
Antibodies Plus C on Metal Stimulation cpm/culture’
Treatmentb
None
None Anti-Thy- 1.2 Anti-L+ 1 Anti-Lyt-2 Anti-L3T4
6,072 + 2,376 2,105 + 1,306 1,105 2 308* 14,325 + 3,893* 2,410+ 827
Pb 30,145 1,556 668 32,884 1,376
+ 4,683 + 162* + 125* 2 7,072 + 260*
Ni 73,928 2,548 2,411 95,543 3,176
f 3,222 31 228* -+ 991* -+ 13,614 f 1,069*
Zn 83,537 5,020 443 101,918 8,837
+ f f + +
9,333 3,330* 251* 11,393 3,953*
Note. Cells were cultured in flat-bottom 96-well plates (5 X lo5 cells/well), in M- 199 containing 5% PBS, and were pulsed and harvested on Day 6. ’ Cultures were stimulated with 100 PM metal in saline. The results represent the means + SD of triphcate cultures and are representative of two separate experiments. b Splenocytes were treated with anti-Thy-l .2 (30-H12), anti-Lyt-1.1 (CE/J anti-CBA/J alloantisera), anti-Lyt-2 (3.155), or anti-L3T4a (GKl.5) as indicated under Materials and Methods. * P < 0.05 relative to respective no-treatment control.
CATIONIC
IMMUNOMODULATION
435
L3T4 plus complement treatment was able to significantly reduce the ability of the metals to induce proliferation. Anti-Lyt-2 plus complement slightly enhanced metalinduced proliferation. Therefore, it appears that T cells bearing L3T4 and Lyt- 1 are required for the initiation of metal-induced proliferation. DISCUSSION The results reported above describe a form of immunomodulation whereby cations can modulate the immune response by activation of T cells. Evidence was presented which suggests that in vitro immunomodulation by the metals Pb, Ni, and Zn is affected by the type of basal medium employed, the presences of exogenous thiols such as 2ME, and the relative levels of cell-cell contact. Furthermore, the ability of the metals to stimulate lymphoproliferation was dependent on the presence of T cells and an Ia+ population of “accessory” cells. Previous work focused on the proliferative effects of these metals occurring in the first 3 days of culture or on the ability of these metals to alter the splenocyte proliferation induced by mitogens such as Con A and LPS on Days l-4 (2,5,6). The “early” proliferative effects have been difficult to consistently reproduce which may be due to changes in media constituents, especially with regard to the source of FBS and to low level endotoxin contamination. Low level endotoxin contamination can affect various in vitro assays and the interpretation of such assays (28). As such, this study has employed media constituents with defined levels of endotoxin contamination which are not immunomodulatory. Under these circumstances these metals do not stimulate “early” proliferation but do stimulate “late” (> Day 5) lymphoproliferation, similar to proliferation induced by soluble antigens or by allogeneic or autologous cells. The type of media employed was demonstrated to affect the ability of these metals to stimulate lymphoproliferation. In studies employing the medium M-199, Zn and Hg were able to induce human lymphocytes to proliferate late (Day 5) in culture ( 13,18). RPM1 1640, M- 199, and MEM were compared as to their abilities to support the metal-induced modulation of the primary in vitro anti-SRBC PFC response and the induction of proliferation by the metals Pb, Ni, and Zn. The media contained the same supplements, including the same amount and lot of FBS; therefore, the differences among the media must lie in some basal media constituent(s). Each medium was able to support the basal PFC and proliferative response; however, the absolute number of the PFCs induced by Pb and Ni was approximately two- to threefold greater in M-199 than in RPM1 1640 or MEM. M-199 also appeared to be the medium of choice in terms of the metal-induced proliferative response. These media differ with respect to to the amounts of GSH, PO:-, Ca*+, Fe*+, and Mg*+ with which the exogeneous metal might interact. Although RPM1 1640 is sometimes considered the medium of choice for lymphocytes (29), the basis for this is not obvious. While we did not perform an exhaustive survey of tissue culture media, it is apparent that medium selection is a critical factor in determining the immunomodulatory activity of these metals. The observed metal response may be considered weak relative to some other forms of lymphostimulation and, therefore, may be particularly sensitive to media effects as is the case with responses directed at minor histocompatibility differences (30). 2ME was shown to enhance metal-induced proliferation regardless of the basal medium employed. Indeed, no proliferation in response to Pb or Zn was
456
WARNER
AND
LAWRENCE
observed in MEM (which contains no GSH) unless 2ME was added. 2ME enhances the viability of cultured cells, is known to enhance the in vitro murine immune response (31), and has been suggested to function by minimizing random disulfide bonding of the surface of murine lymphocyte, thereby allowing the proper disulfides to form when the appropriate stimulus is received (32). These metals are capable of interacting with free sullhydryls (33) and may interact with specific cellular thiols that are involved in cellular triggering events. Cell density (cells per well) was determined to be an important factor in demonstrating metal stimulation of lymphocyte proliferation. For each of the metals tested a sharp decrease in the response was observed at densities between 1 X lo5 and 2 X lo5 cells per well in flat-bottom wells. The decrease in the response associated with decreased cell density was gradual in round-bottom wells which promote cell-cell contact, suggesting that cell-cell contact is important in the induction of proliferation by these metals. It is interesting to note that the magnitude of the response was lower in the round-bottom culture, suggesting that the extent of activation may be down regulated by excessive cell-cell interactions such as lymphocyte-macrophage interactions (34). Previous attempts to determine the nature of metal-induced immunomodulation have focused on the ability of the metal to alter responsiveness induced by T-cell or B-cell mitogens. Pb and Ni were shown to enhance the proliferative response induced by LPS which was taken to indicate that Pb and Ni acted primarily on B cells (5, 6, 20). Other investigators demonstrated that Pb alters Con A-induced proliferation while having little effect on B-cell mitogen responses (21), and as such primarily altered T-cell functions. Zn was shown to be essential for lymphocyte mitogenesis (18, 35) and was shown to enhance proliferation of spleen cells from C57Bl/6 mice stimulated with supraoptimal doses of Con A (36). None of the metals tested herein significantly affected the ability of Con A or LPS to induce lymphocyte proliferation. B cells and T cells were isolated and examined for their ability to initiate metal-induced lymphoproliferation. T-cell preparations proliferated as well as unselected splenocytes in response to metal stimulations, whereas B-cell preparations were not induced to proliferate by the metals. The possibility that the observed proliferation was due to contaminating cells does not seem likely in view of the initial purity of the respective populations and the magnitude of the response. Anti-la plus complement treatment of splenocytes completely ablated the metal-induced proliferative response. The T-cell preparations employed in these studies were not depleted of macrophages and as such contained Ia + “accessory” cells. Anti-Ia plus complement treatment of T-cell preparations also abrogated metal-induced proliferation. Clearly T cells and Ia+ accessory cells are important in metal-induced lymphoproliferation. As indicated in Table 5 a Thy-1.2+, Lyt-l+, Lyt-2-, L3T4+ cell appears to be necessary in order for the metals to stimulate proliferation. Anti-Lyt-2 plus complement treatment consistently, but not significantly, enhanced the proliferative response, suggesting that Lyt-2+ cells may be capable of slightly suppressing the response, including the background autologous response. Cations are involved in a variety of important biochemical pathways. Zn is known to be a cofactor for DNA polymerase I as well as a number of proteases (37). It has been suggested that exogeneous Zn may enhance the activity of DNA polymerase and thereby enhance [3H]thymidine incorporation (38). The fact that B cells and T cells differ in their ability to be stimulated by Zn argues against this hypothesis. Ion
CATIONIC
IMMUNOMODULATION
431
fluxes (Ca’+, Naf, K+, etc.) across the plasma membrane are thought to be intimately involved with the regulation of growth and transmembrane signaling in general (39,40). Preliminary studies in this laboratory using Ca2’ channel blockers suggest that Pb does not require active Ca2’ channels in order to modulate the in vitro PFC response (data not shown). The relative toxicity of these metals has been hypothesized to be due to their abilities to interfere with intracellular biochemical pathways which require Ca2+, especially those involving calmodulin (41). Choa et al. (42) demonstrated that Pb and Zn were able to enhance the ability of calmodulin to stimulate phosphodiesterase activity; however, Ni had little effect on calmodulin-induced phosphodiesterase activity. Studies in this laboratory demonstrated that Ni but not Pb nor Zn was able to restore the in vitro PFC in cultures chelated with EGTA (5). Ni itself is known to be a potent Ca2’ channel blocker (43). Presently, there does not appear to be a general biochemical explanation for the ability of all three metals to stimulate lymphoproliferation. Metal-induced lymphoproliferation requires the presence of both T cells and Ia+ cells, suggesting several interesting hypotheses which may account for the immunomodulatory effects of these metals on lymphoproliferation as well as the in vitro PFC response. The metals may alter the conformation of class II molecules on the surface of Ia+ cells. T cells specific for this metal-induced alteration of self would become activated in a manner analogous to the well-defined system of hapten-modified self (44), drug-modified self (45), or metal-modified self (22, 23). T cells responsive to altered self are known to exert both positive and negative effects on the in vitro PFC response (46) which could account for the observed differential effects of these metals on the in vitro PFC response. Irradiation (1000 rad) of T cells prior to culture in the presence of normal syngeneic B cells results in a significant increase in the metal (including Zn)-enhanced PFC response (G. L. Warner and D. A. Lawrence, work in progress), suggesting the presence of metal-activated radiosensitive suppressor T cells as described previously in a variety of systems (46-48). It should be noted that the ability of these metals to stimulate proliferation or modulate the in vitro PFC response is not peculiar to the CBA/J (H-23 strain of mouse since we have observed the phenomena using BALB/c (H-2d), DBA/2 (H-2”) and C57B1/6 (H-26) strains of mice (data not shown). The autologous mixed-lymphocyte response (AMLR) has recently gained much attention as a property of the normal immune response (49). As in stimulation by hapten-modified self, the autologous mixed-lymphocyte response is known to be capable of generating both helper and suppressor cells (50, 5 I), and agents which are known to enhance the expression of Ia molecules on B cells are thought to augment the AMLR (52). It is possible that the metals act directly or indirectly to enhance the expression of Ia, thereby enhancing the magnitude of the AMLR. Metallothioneins are a family of proteins rich in cysteine which are inducible by heavy metals, glucocorticoids, IFN, and IL- l(53). The metallothioneins have been thought to be primarily involved with heavy metal detoxification; however, it has recently been suggested that their primary function may be as a hydroxyl radical scavenger necessary to protect cells from oxidative insults generated by activated macrophages (54). This would suggest that IFN, IL- 1, and metallothionein are coordinately regulated. In light of the above results it would be interesting to know whether metal inducers of metallothionein are capable of inducing the expression of IFN and/or IL-l. If so, the metalinduced IFN could then induce an increase in Ia expression, thereby augmenting
438
WARNER
AND LAWRENCE
the AMLR. The possibility of metal induction of IFN could also account for the augmentation of the PFC response since (y)IFN is thought to have at least some Tcell replacing factor (TRF) activity (55). It is interesting to note that Ni and Pb enhance the SRBC-specific PFC response of T-cell-depleted splenocytes (6), a phenomenon associated with TRF activity. In conclusion, these results indicate that Pb, Ni, and Zn are capable of modulating the immune response in vitro. The possibility that these metals modulate the immune response by altering self, upsetting a normal immunohomeostatic regulatory mechanism involving T-cell recognition of self and/or affecting the production/expression of various lymphokines, is intriguing. Cation modulation of the immune response could serve as a model system for observing effects on the immune response at the cellular, biochemical, and genetic levels. REFERENCES 1. Graham, J. A., Miller, F. J., Daniels, M. J., Payne, E. A., and Gardner, D. E., Environ. Res. 16, 77, 1978. 2. Blakley, B. R., and Archer, D. L., Toxicol. Appl. Pharmacol. 61, 18, 198 I. 3. Lawrence, D. A., Infect. Zmmun. 31, 136, 1981. 4. Malave, I., and Benaim, I. R., Cell. Zmmunol. 89,322, 1984. 5. Lawrence, D. A., Toxicol. Appl. Pharmacol. 57,439, 198 1. 6. Lawrence, D. A., Znt. J. Zmmunopharmacol. 3,153, 198 1. 7. Underwood, E. J., In “Trace Elements in Human Health and Disease” (A. S. Prasad and D. Oderleas, Eds.), Vol. 2, pp. 269-280. Academic Press, New York, 1974. 8. Prasad, A. S., Nutr. Rev. 41, 197, 1983. 9. Sirover, M. A., and Loeb, L. A., Science 194, 1434, 1976. 10. Babich, H., Devanas, M. A., and Stotzky, G., Environ. Res. 37,253, 1985. 11. Cronin, E., “Contact Dermititis.” Churchill Livingstone, New York, 1980. 12. Nordlind, K., Znt. Arch. Allergy Appl. Zmmunol. 75,333, 1984. 13. Berger, N. A., and Skinner, A. M., J. Cell. Eiol. 61,45, 1974. 14. Ruhl, H., Kirchner, H., and Buchert, G., Proc. Sot. Exp. Biol. Med. 137, 1089, 197 1. 15. Nordlind, K., and Hem%, A., Znt. Arch. Allergy Appl. Zmmunol. 75,330, 1984. 16. Al-Tawil, N., Marcusson, J. A., and Moller, E., Acta Derm. Venereal. 61,5 11, 1981. 17. Nordlind, K., Znt. Arch. Allergy Appl. Zmmunol. 73, I5 1, 1984. 18. Alford, R. H., J. Zmmunol. 104,698, 1970. 19. Bendtzen, K., and Maryland, L., Scund. J. Zmmunol. 15,81, 1982. 20. Shenker, B. J., Matarazzo, W. J., Hirsch, R. L., and Gray, I., Cell. Zmmunol. 37, 19, 1977. 21. Blakley, B. R., and Archer, D. L., Toxicol. Appl. Pharmacol. 62,183,1982. 22. Pelletier, L., Pasquier, R., Hirsch, F., Sapin, C., and Druet, P., Eur. J Zmmunol. 15,460, 1985. 23. Sin&ha, F., Scheidegger, D., Garotta, G., Scheper, R., Pletscher, M., and Lanzavecchia, A., J. Zmmunol. 135,3929, 1985. 24. Lawrence, D. A,, and Weigle, W. O., J. Exp. Med. 139,943,1974. 25. Jerne, N. K., and Nordin, A. A., Science 140,405,1963. 26. Wysocki, L. J., and Sato, V. L., Proc. Natl. Acad. Sci. USA 75,2844, 1978. 27. Davidson, W. F., and Parish, C. R., J. Zmmunol. Methods. 7,29 1, 1975. 28. Eastman, A. Y., and Lawrence, D. A., Stand. J. Zmmunol. 21,35,1985. 29. Moore, G. E., Ge.mer, R. E., and Franklin, H. A., J. Amer. Med. Assoc. 199,5 19, 1967. 30. Click, R. E., Adelmann, A. M., and Azar, M. M., J. Zmmunol. 134,2948, 1985. 31. Chen, C., and Hirsch, J. G., .Z.Exp. Med. 136,604,1972. 32. Noelle, R. J., and Lawrence, D. A., Cell. Zmmunol. 60,453, 198 1. 33. Passow, H., In “Effects of Heavy Metals on Cells, Subcelhnar Elements, and Macromolecules” (J. Maniloff, L. Coleman, and M. Miller, Eds.), pp. 291-340. Thomas, Springfield, Ill., 1970. 34. Metzger, Z., Hoffeld, J. T., and Oppenheim, J. J., J. Zmmunol. 124,983,1980. 35. Zanzonico, D., Femandes, G., and Good, R. A., Cell. Zmmunol. 60,203, 198 1. 36. Malave, I., and Rondon, I., Cell. Zmmunol. 89,322, 1984.
CATIONIC 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 41. 48. 49. 50. 5 1. 52.
IMMUNOMODULATION
Parisi, A. F., and Vallee, B. L., Amer. J. Clin. Nutr. 22, 1222, 1969. Williams, R. O., and Loeb, L. A., J. Cell. Biol. 58,594, 1978. Rosoff, P. M., and Cantley, L. C., Proc. Natl. Acad. Sci. USA 80,1541, 1983. Lichtman, A. H., Segal, G. B., and Lichtman, M. A., Blood61,413,1983. Cox, J. L., and Harrison, S. D., Biochem. Biophys. Res. Commun. 115,106,1983. Chao, S. H., Sazuki, Y., Zysk, J., and Cheung, W. Y., Mol. Pharmacol. 26,15, 1984. Hagiwara, S., and Byerly, L., Annu. Rev. Neurosci. 4,69, 1981. Shearer, G. M., Lozner, E. C., Rehn, T. G., and Schmitt-Verhulst, A. M., J. Exp. Med. 141,930,1975. Nagata, N., Hurtenbach, U., and Gleichmann, E., J. Immunol. 136,136,1986. Eastman, A. Y., and Lawrence, D. A., J. Immunol. 128,926, 1982. Swain, S. L., and Dutton, R. W., J. Immunol. 118,2262, 1971. Basten, A., Miller, J. F. A. P., and Johnson, P., Transplant. Rev. 26, 130, 1975. Finnegan, A., and Hodes, R. J., J. Immunol. 136,793, 1986. Yu, D. T. Y., Chiorazzi, N., and Kunkel, H. G., Cell. Immunol. 50,305, 1980. Smith, J. B., and Knowlton, R. P., J. Immunol. 123,419, 1979. Crow, M. K., and Kunkel, H. G., Cell. Immunol. 90,555, 1985. 53. Karin, M., CeN41,9, 1985. 54. Karin, M., Imbra, R. J., Heguy, A., and Wong, G., Mol. Cell. Biol. 5,2866, 1985. 55. Brunswick, M., and Lake, P., J. Exp. Med. 161,953, 1985.
439
101,425-439 (1986)
IMMUNOLOGY
Stimulation
of Murine Lymphocyte
GARVIN L. WARNERANDDAVID
Responses
by Cations’
A. LAWRENCE
Department of Microbiology and Immunology, Neil Hellman Medical Research Building, Albany Medical College of Union University, Albany, New York 12208 Received February 13, 1986; accepted May 5, 1986 The capacity of the heavy cations Pb, Ni, and Zn to modulate murine in vitro lymphocyte responses was examined. Pb and Ni ( 100 @4) were shown to enhance the in vitro plaque-forming cell (PFC) response to sheep red blood cells while 100 &f Zn had inhibitory effects.Each metal was able to stimulate the proliferation of murine splenocytes as determined by [‘H]thymidine incorporation and autoradiography. The enhancing effect of the metals on the PFC response was observed whether the results were expressed on a per culture or a per cell basis, indicating an actual increase.in B-cell differentiation. Both the PFC response and the proliferative response were shown to be sensitive to the type of medium employed (M- 199 gave optimum results) and to the presence or absence of 2-mercaptoethanol. As in autologous mixed-lymphocyte responses peak proliferation occurred after Day 5 in culture, was cell density dependent, and required the presence of both T cells and Ia+ cells. Treatment of splenocytes with anti-Thy-l .2, anti-Lyt- I, or anti-L3T4 plus complement completely abrogated the proliferative response, indicating that a Lyt-l+, Lyt-2-, L3T4+ T-cell was required for the induction of proliferation. The data are consistent with the hypothesis that the metals are capable of modifying the immune response directed at self either by directly altering self constituents (class II) or by modulating the autologous T-cell response. 0 1986 Academic press, Inc.
INTRODUCTION The heavy cations Pb, Ni, and Zn have been shown to modulate immune responses and in vitro (4-6). Although Zn and Ni (in rats) are essential trace elements (7) and Zn deficiency causes severe immune abnormalities (8), these metals have been suggested to be carcinogens (9, 10). Furthermore, they have been shown to elicit contact dermititis reactions (11) presumably by interacting with and altering self constituents and rendering them immunogenic. As such, the association of these metals with host constituents has important health implications. The mechanism whereby these metals exert their immunomodulatory effects is not known. Ni and Zn stimulate DNA synthesis of human peripheral blood lymphocytes ( 12- 14). Ni also is known to stimulate DNA synthesis of human thymocytes ( 15). The degree of Ni stimulation has been shown to correlate ( 16) with the Ni sensitivity of individuals although some overlap between control and N&sensitive patients has been observed ( 17). In addition to the mitogenic activities ofNi and Zn, these metals have been shown to restore the proliferative response of lymphocytes exposed to the metal chelain vivo (l-3)
’ This work was supported by NIH Grant ES 03 179.
425 0008-8749/86$3.00 copyright 8 1986 by Academic I’res, Inc. AU rights of reproduction in any fom reserved.
426
WARNER
AND
LAWRENCE
tors EDTA (18) and phenanthroline ( 19). Pb, Ni, and Zn induce murine splenocyte DNA synthesis (5,6,20). Pb and Ni have been demonstrated to enhance DNA synthesis induced by the B-cell mitogen LPS* (5,6), while Pb has been reported to enhance Con A- and PHA-induced T-cell proliferation (2 1). In terms of functional immunity, Pb and Ni have been shown to enhance in vitro humoral immune responses, while Zn was inhibitory (5). Furthermore, the enhancement of the in vitro PFC response by Pb occurred in the absence of T cells (6). It appears that these metals have pleiotropic effects and are capable of modulating both T-cell and B-cell activities. The ability to discriminate between self and nonself is central to the role of the immune system. As such, the immune system is delicately balanced between positive and negative regulatory influences. Agents which have the capacity to upset this delicate balance may affect the health of the host by decreasing resistance to pathologic agents or by contributing to the generation of autoimmune disease. In a recent report by Pelletier et al. (22) it was demonstrated that HgCl, treatment of the Brown-Norway rat altered resident lymphocytes in such a manner that they were able to stimulate in vivo lymphocyte proliferation when transferred to an autologous host, the hypothesis being that the metals interact with and alter self constituents, thereby rendering these self antigens immunogenic. Ni-specific T-cell clones were recently isolated from peripheral blood of Ni contact-sensitive patients (23); these T-cell clones responded to Ni in an MHC class II-restricted fashion. Hg is quite toxic in the mouse and displays none of the immunomodulatory activities ascribed to Pb, Ni, and Zn (5); however, the effect of Hg in the rat and Ni in the human may serve as models whereby some of the immunomodulatory activities of Pb, Ni, and Zn in the mouse may be explained. In this investigation, the in vitro effects of Pb, Ni, and Zn on murine splenocyte proliferation have been further defined and examined. Different tissue culture media were compared for their ability to sustain metal-induced modulation of both in vitro PFC response and proliferation, and the requirement for 2-mercaptoethanol(2ME) was determined. The kinetics of lymphoproliferation was measured for extended culture periods. Peak responses were observed late (Days 5-7). Cellular contact was also implicated as being important in the overall response mediated by these metals. Lymphocyte subset depletion experiments were performed to determine whether T cells, B cells, and/or IaC cells were required for these metals to induce proliferation. The results presented herein are consistent with the hypothesis that Pb, Ni, and Zn modulate the in vitro murine immune response by altering the immunogenicity of self, possibly by modifying class II MHC molecules. MATERIALS
AND METHODS
Animals. Female CBA/J mice (Jackson Laboratory, Bar Harbor, Maine) 2-4 months of age were used for all studies. Mice were maintained on laboratory chow and acidified, chlorinated water, pH 3.0, ad libitum. Media and reagents.RPMI-1640, M- 199 (with Hanks BSS), and MEM were purchased from M. A. Bioproducts (Walkersville, Md.) and were supplemented with 5% * Abbreviations used: 2ME, 2-mercaptoethanol; PBS, phosphate-buffered saline; BSS, balanced salt solution; Con A, concanvalin A, LPS, lipopolysaccharide; MHC, major histocompatability complex; PFC, plaque-forming cell; SRBC, sheep red blood cell; FBS, fetal bovine serum; GSH, glutathione; AMLR, autologous mixed-lymphocyte response; IFN, interferon; IG 1, interleukin 1; TRF, T-cell replacing factor.
CATIONIC
IMMUNOMODULATION
427
FBS (co.8 rig/ml endotoxin; Sterile Systems, Logan, Utah), L-glutamine (1 mM), Napyruvate (1 mM), nonessential amino acids (0.1 m&Q, gentamicin (50 pg/ml), and NaHC03. 2ME (5 X 10e5 A& Eastman Kodak, Rochester, N.Y.) was included in all media unless otherwise indicated. Stock solutions of PbCl*, NiCl*, and ZnClz (Fisher Scientific, Rochester, N.Y.) were prepared in physiologic saline at a concentration of 1 x 10m2 M. Sheep red blood cells (SRBC) were purchased from Colorado Serum (Boulder, Colo.). Monoclonal antibodies. The following hybridoma cell lines were obtained from the American Type Culture Collection (Rockville, Md.): 30-H 12 (anti-Thy- 1.2, rat IgG), 3.115 (anti-Lyt-2, rat IgM), GK1.5 (anti-L3T4a, rat IgG), and HO-13-4 (anti-Thy1.2, mouse IgM). Cells were cultured in the recommended media, and the supernatants harvested 7 days following entry into stationary phase and dialyzed into phosphate-buffered saline (PBS, pH 7.4). Each preparation was titered against thymocytes using rabbit complement and found to have a 50% cytotoxic titer greater than 1: 1000. For depletion experiments the rat monoclonal reagents were used at a final dilution of 1: 10. The supematant from HO-l 3-4 was precipitated with 50% ammonium sulphate and reconstituted to 10% of its original volume with PBS. Primary in vitro PFC assay, Spleens were aseptically removed and a single cell suspension was made by pressing the spleens between the frosted ends of two microscope slides. Clumps were allowed to settle and cells were cultured as described (24). Briefly, 5 X lo6 spleen cells/O.5 ml, plus or minus antigen (SRBC), plus or minus metal, were cultured for 5 days in special gas composed of 10% C02, 7% 02, 83% N2. On Day 5, cells of triplicate cultures were harvested and pooled and the number of plaque-forming cells (PFC) was determined by use of a modification of the Jeme plaque assay (25). Only direct plaques were detected. Proliferative assay. Metal stocks were appropriately diluted with saline and 25 ~1 was added to the appropriate wells of 96-well flat-bottom tissue or round-bottom culture plates (catalogue No. 3596 or 3799, respectively; Costar, Cambridge, Mass.), such that the final concentration of metal was between 5 and 100 PM, as indicated in the text. Zero metal controls received saline alone. A single cell spleen cell suspension (0.2 ml) was added such that each well contained 4 X lo4 to 8 X lo5 cells/well. Cells were cultured in special gas composed of 10% C02, 7% 02, 83% N2. On the day of harvest 0.5 &i of [3H]thymidine (New England Nuclear Corp.), 25 ~1, was added and individual wells were harvested 6 hr later using a multiple cell culture harvester (Skatron AS, Liebgen, Norway). Filter disks were dried and counted in a Nuclear Chicago Isocap 300. Lymphocyte subpopulation separations. T cells were isolated by panning on antimouse immunoglobulin-coated plates as previously described (26). Briefly, viable splenocytes were isolated on a Ficoll-metrizoate gradient (density 1.090) (27) and suspended in PBS containing 10% heat-inactivated FBS and 0.1% NaN3 at a concentration of 10’ cells/ml; 3 ml of this suspension was placed on an 18-cm bacteriologic petri dish that previously had been coated with rabbit F(ab’)2 anti-mouse immunoglobulin. The unbound cells were gently collected and the plates gently washed once with PBS to maximize recovery. The cells were then washed 3~ with BSS (balanced salt solution, pH 7.4) containing 2% FBS. B cells were obtained from animals that had been injected with 50 ~1 anti-mouse thymocyte serum (M.A. Bioproducts) 48 hr prior to exsanguination. A single cell suspension of splenocytes was obtained, resuspended at l-4 X 10’ cells/ml in BSS,
428
WARNER
AND LAWRENCE
2.5 9 $ x
2
2
1.5
2 3 2
1
y
0.5
5#n8
Pb
*
B
1. Modulation of the in vitro PFC response by Pb, Ni, and Zn: comparison of MEM (dotted), M- 199 (slashed), and RPMI-1640 (cross-hatched). Splenocytes (5 X lo6 cells/well) were cultured in the presence or absence of 1 X 1Oe4M Pb, Ni, or Zn as indicated. On Day 5 cells were harvested and assayed for PFCs as described under Materials and Methods. The data presented are. representative of three separate experiments. FIG.
containing 2% FBS, and subjected to two cycles of anti-Thy- 1.2 ( 1:25; HO- 13-4) and anti-Lyt- 1.1 ( 1: 125; CE/J anti-CBA/J alloantiserum; 30 min, 4”C), plus rabbit complement (1:25; 37°C 45 min). The cells were spun through 100% FBS and washed 3X with BSS containing 2% FBS. All other antibody plus complement treatments were performed using a one-cycle protocol as follows. Antibodies were used at a final dilution of 1: 10 except for antiIak (A.TH anti-A.TL alloantiserum) and anti-Lyt- 1.1 (CE/J anti-CBA/J alloantisera) which was used at a fin&lilution of 1:25. Cells were incubated with antibody for 30 min, 4°C pelleted, and resuspended in rabbit complement (1:25) for 45 min, 37°C. The cells were then centrifuged through 100% FBS and washed 3X with BSS containing 2% FBS. Autorudiogruphy. Cells were cultured and pulsed as in the proliferative assay. Cells were harvested into 1.5-ml polypropylene microcentrifuge tubes, centrifuged through 100% FBS, washed 2X with PBS, and cytocentrifuged onto clean glass slides. The slides were then air dried, fixed with 1% gluteraldehyde, and emulsed using NTB-2 (Eastman Kodak). The slides were exposed for 1 week at 4°C developed using developer D-19, fixed with a 25% solution of Na-thiosulfate and stained with Wright’s stain. Labeled nuclei were counted by light microscopy under oil. Statistical analysis. Results are expressed as means + SD. The difference between means was assessed by the two-tailed Student t test. Differences were considered significant when P was greater than 0.05. RESULTS Characterization
of the in Vitro Primary
Response
The ability of RPMI-1640, M-199, and MEM to sustain an in vitro primary immune response was determined. Each basal medium was supplemented equivalently as described under Materials and Methods. As demonstrated in Fig. 1, the enhancing effects of Ni and Pb were observed regardless of the medium employed; however, the degree of metal enhancement was significantly greater in M- 199 compared with either RPMI-1640 or MEM. The inhibition exhibited by Zn was apparent in each medium as well. M-199 appears to be the medium of choice when observing metal-
CATIONIC
429
IMMUNOMODULATION TABLE 1
Effect of Metals on Cell Recovery and the in Vitro PFC Response Metal 1x 5x 1X 5X 1X 5X
0 10-4MPb lO-5 MPb 10e4MNi IO-‘MNi 10m4MZn 10e5MZn
Cell recovery’l (X106) 6.42 7.51 5.50 8.40 6.61 10.70 6.90
PFC/cuIture 640+23 2080 + 224* 1756 f 289* 1320 f 100* 716k33 289 f 73* 577 + 76
(l.OO)b (3.25) (2.74) (2.06) (1.12) (0.45) (0.90)
PFC/ lo6 cells 299.1 830.9 957.8 471.4 325.0 81.1 251.0
f 11 f 89* 2 158* f 36* f 15 f 20* + 33
(1.00) (2.78) (3.20) (1.58) (1.09) (0.27) (0.84)
Note. CBA/J splenocytes were cultured in M- 199 containing 5% FBS at a density of 5 X lo6 cells/O.5 ml. After 5 days in culture triplicate wells were pooled and the number of direct PFCs enumerated. a Cell counts were made on pooled triplicate wells and are expressed as total number of cells recovered per three wells. b Number in parentheses indicates stimulation index (metal treated/O metal control). * P < 0.05 relative to 0 metal control.
induced immunomodulation of the in vitro PFC response. It should be noted that there were no differences in either cellular viability or cell recovery among the various media (data not shown). Each of the metals tested caused an increase in the number of recoverable cells compared with the saline controls (Table 1). Even though Zn inhibited the PFC response, it enhanced cell recovery. The enhanced PFC response by Pb and Ni was observed regardless of whether the PFC results were expressed on a per culture or a per cell basis (Table l), indicating that the enhancement was due to an actual increase in the number of differentiated B cells rather than solely due to enhanced recovery. Pb increased the PFC response in the absence of SRBC; however, as previously reported (5), the magnitude of the enhancement of the specific response was always greater than the nonspecific response.
Characterization of Metal-Induced Lymphocyte Proliferation The ability of RPMI- 1640, M- 199, and MEM to support metal-induced lymphocyte proliferation was assessed. Spleen cells were stimulated with optimal metal concentrations and were pulsed and harvested on Day 6. As demonstrated in Fig. 2, Ni was capable of inducing significant [3H]thymidine incorporation regardless of the basal medium employed. Pb and Zn did not induce significant [3H]thymidine incorporation in MEM without 2ME (Fig. 2A). It should be noted that the absence of a response could not be accounted for by a shift in kinetics (data not shown). Regardless of the basal medium employed, the proliferative response induced by each of the metals was greater in the presence of 2ME (Fig. 2B). M-199 consistently supported metal-induced responses which were significantly greater than the response observed with MEM. M- 199 supported a significantly greater Zn-induced response compared with RPMI-1640 while the difference between M-199 and RPMI-1640 with respect to Pb and Ni stimulation was not significant; however, M- 199 did consistently produce a higher proliferative response regardless of the medium employed. In order to be sure that we were indeed looking at [3H]thymidine incorporation into nuclear
430
WARNER
AND LAWRENCE
FIG. 2. Metal induction of lymphocyte proliferation, comparison of MEM (dotted), M-199 (slashed), and RPMI- 1640 (cross-hatched). Splenocytes (5 X 10’ cells/well) were cultured in the presence or absence of 1 X 10e4 M Pb, Ni, or Zn as indicated. On Day 5 the wells were pulsed with 0.5 pCi of [3H]thymidine for 6 hr, harvested onto glass fiber mats, dried, and counted by liquid scintillation spectroscopy. Responses were assayed in the absence (A) or presence (B) of 5 X IO-’ A4 2ME. The data presented are representative of three separate experiments.
DNA, autoradiography of whole cells pulsed with [3H]thymidine was performed. As indicated in Table 2, Pb, Ni, and Zn each caused an increase in the percentage of labeled nuclei, and this increase corresponded to the relative degree of thymidine incorporation assessed by liquid scintillation counting. In addition, cell viability in the presence of metal was equal to or greater than the control group. Taken together these data demonstrate that the metals Pb, Ni, and Zn induce murine lymphocytes to initiate nuclear DNA synthesis late in culture. TABLE 2 Effect of Metals on Viability and [3H]Thymidine
Incorporation into Nuclear DNA
% Viable“
% Labeled nucleib
61 15 81 59 82 65 64
12 20 ND’ 33 ND 36 ND
Metal 1x 5x 1X 5X 1X 5X
0 10-4MPb lo-‘MPb 10m4MNi IO-‘MNi lo-‘+MZn lo-‘MZn
Note. CBA splenocytes were cultured in the presence or absence of metal at a density of 5 X 10’ cells/ well. On Day 6 individual wells were pulsed with 0.5 &i [‘H]thymidine for 6 hr and harvested into polypropylene tubes. ’ Viability was assessedby exclusion of trypan blue. b Cells were cytocentrifuged onto clean glass slides, fixed, stained, and emulsed with NTB-2. Slides were developed 7 days later and labeled nuclei counted by light microscopy. ’ ND indicates that value was not determined.
CATIONIC
431
IMMUNOMODULATION
120 n k
I#) m 60
2x 2s 9 8" 20 0
LPS
Con
A
3. Effect of metals on mitogen-induced lymphocyte proliferation. Splenocytes (2 X 10’ cells/well) were cultured in the presence of LPS (10 &ml) or Con A (2.5 &ml) as indicated. Cells were also stimulated with 0 (open), 1 X 10m4M Pb (slashed), 1 X 10m4M Ni (cross-hatched), or 1 X 10m4M Zn (closed). On Day 2 individual wells were pulsed with 0.5 &i of [‘Hlthymidine for 6 hr, harvested onto glass fiber mats, dried, and counted by liquid scintillation spectroscopy. The data presented are representative of three separate experiments. FIG.
Eflect of Heavy Metals on Con A- and LPS-Induced Mitogenesis In order to determine whether Pb, Ni, and Zn had a generalized capacity to modulate lymphocyte proliferation, we examined the ability of these metals to alter mitogen-induced proliferation. Figure 3 demonstrates that Pb, Ni, and Zn did not significantly enhance nor inhibit LPS- or Con A-induced proliferation.
Kinetics of Metal-Induced Proliferation Earlier studies (6) had indicated that Pb caused an early (Day 3) increase in [3H]thymidine incorporation. This early proliferative effect has been difficult to repeat consistently and probably was not due to Pb alone, in that it was dependent on the lot of FBS used (28). Pb enhancement of the PFC response has been consistent and is assayed for relatively late in culture (Day 5). Figure 4 shows the extended kinetics of metal-induced [3H]thymidine uptake by cultured mu&e splenocytes covering Days 2-9. Four doses of metal were employed in these studies, 1 X lop4 A4, 5 X 10F5 M, 1 X IO-’ M, and 5 X lop6 M, except in the case of Zn where previous work had demonstrated that concentrations of less than 1 X 1Oe5M had no effect. As shown in Figure 4A, Pb caused a dose-dependent proliferative response which peaked on Day 7 at approximately 90,000 cpm with a Pb dose of 1 X lop4 M. Ni (Fig. 4B) showed a dose-related response which peaked on Day 6 or 7 ( 1 X 10e4 It& 100,000 cpm). Zn (Fig. 4C) induction of the proliferative response occurred earliest (100,000 cpm on Day 6 with a dose of 1 X 10e4 M). The induction of proliferation by Zn always occurred earlier than that by Pb or Ni. Pb-induced proliferation sometimes peaked on Day 6 rather than Day 7 and usually was less than that of Ni and Zn in terms of the absolute number of cpm. The subtle variations in kinetics may have been due to minor variations in the final metal concentration or to minor variations in the final number of cells per well. Visual examination of the cultures revealed the appearance of small clusters of cells, approximately 3 days postinitiation of culture, the number of which appeared to correlate with intensity of the [3H]thymidine incorporation. The kinetics of the response are similar to other in vitro immune responses
432
WARNER
AND LAWRENCE
50
10 5
100 x
2 c3 3 P if!
50
10 100
50
10 2
3
4
5 6 OAYSlNCUlTURE
7
6
9
FIG. 4. Metal induction of lymphocyte proliferation: extended kinetics. Splenocytes (5 X lo5 cells/well) were cultured in complete M-199 containing 5 X lo-’ MZME in the presence of 0 (open squares), 1 X 10e4 M (closed squares), 5 X 1Oe5M (closed triangles), 1 X I Om5M (closed circles), or 5 X 10m6M (open circles) Pb (A), Ni (B), or Zn (C). On the indicated days the wells were pulsed with 0.5 pCi of [3H]thymidine for 6 hr, harvested onto glass fiber mats, dried, and counted by liquid scintillation spectroscopy. The data presented are representative of three separate experiments.
which require cell-cell contact such as the autologous or allogeneic mixed-lymphocyte responses.
Eflect of Cell Density on Metal-Induced Mitogenesis The influence of cell-cell interaction on the ability of these metals to induce lymphocyte proliferation was examined. Splenocytes placed in flat-bottom wells settle out randomly and are relatively evenly dispersed within the well. Round-bottom wells allow for the settling of the cells into the center of the well forming a relatively tight “button.” If cell-cell interactions are important in order to initiate the response, then as the cells become more dilute (sparse) in the flat-bottom wells a critical density is reached where the response drops off dramatically. In the round-bottom wells, the geometry of the well maximizes cell-cell contact; thus the response should fall off gradually, directly proportional to the number of cells per well. Figure 5 demonstrates the results of one such cell density experiment comparing round- and flat-bottom wells. Splenocytes were cultured at densities ranging from 8 X 1O5to 4 X 1O4cells per well. The response in flat-bottom wells dropped off markedly at approximately 1
CATIONIC
00
433
IMMUNOMODULATION
40
20 CELLS/WELL
10
S
4
x 1O‘4
5. Effect of cell density on cation-induced proliferation. Splenocytes were cultured in M-199 at densities ranging from 4 X lo4 to 8 X lo4 cells/well in either round-bottom [closed circles] or flat-bottom [closed squares] wells. Individual wells were stimulated with 100 pM Pb (A), Ni (B), or Zn (C). Cultures were pulsed with 0.5 #Ji of [3H]thymidine on Day 6 and harvested 6 hr later. The data presented are representative of three separate experiments. FIG.
X lo5 cells per well in the case of Ni and at approximately 2 X lo5 cells per well for both Pb and Zn. The decreased response in the round bottom wells is clearly more gradual. The peak [3H]thymidine incorporation was less in round-bottom wells than in flat-bottom wells; however, the kinetics of the response did not appear to change with regard to the type of plate used (data not shown).
Subpopulations
of Lymphocytes Requiredfor
the Proliferative
Response
The ability of Pb, Ni, and Zn to induce the proliferation of T cells, B cells, or Iacells was investigated. B cells were prepared by vigorous depletion of T cells, utilizing a combined treatment of in vivo rabbit anti-mouse thymocyte serum and two in vitro cycles of antibody plus rabbit complement as described under Materials and Methods. T cells were prepared by panning on anti-mouse Ig-coated petri plates as described by Wysocki and Sato (26). These preparations routinely contained fewer than 2% contaminating cells and often contained undetectable numbers of the eliminated population of cells as determined by flow cytofluorometry. As shown in Table 3, the B-cell preparations failed to proliferate in response to Pb, Ni, or Zn, whereas the T-cell preparations proliferated in response to the metals.
434
WARNER
AND LAWRBNCE TABLE 3
Metal Stimulation of Lymphocyte Subpopulations cpm/cultun? Cell preparations0
None
Pb
Unseparated T cells B cells T cells + B cells Anti-Ia-treated T cells Anti-Ia-treated splenocytes
8055 + 1404 705922519 2025 1 407 50582 126 169+ 43* 26lk 57”
35,392 + 66,682 k 918k 19,195 f 16Ok 206+
8725 6673* 128* 8824 19* 64*
Ni
Zn
7 1,932 +- 4960 84,466 + 5868* 1,390 + 137* 64,926 + 1194 1,068 + 720* 269+ 86;
48,459 + 6565 90,691 I!Z9238” 535* 133* 42,065 +- 4162 784+ 325* 134+ 85*
Note. Cells were cultured in flat-bottom 96-well plates (5 X IO5 cells/well), in M-199 containing 5% PBS, and were pulsed and harvested on Day 6. Cultures were stimulated with 100 &metal in saline. a T cells were obtained by depletion of B cells on anti-mouse Ig-coated plates. B cells were obtained from animals that were injected with ATS 48 hr prior to exsanguination and were subjected to two cycles of anti-Thy- 1.2 and anti-Lyt- 1.2 plus complement treatment. T cells and B cells were mixed at a ratio of 1: 1. Ia+ cells were depleted by one cycle of anti-Ia (A.TH anti-A.TL alloantisera) plus complement treatment. b The results represent the means of triplicate cultures + SD and are representative of three separate experiments. * P < 0.05 relative to respective unseparated control.
Clearly, T cells are required for the induction of proliferation by Pb, Ni, and Zn. The ability of anti-Ia treatment to abrogate the metal-induced proliferative response was also examined. As demonstrated in Table 3, treatment of either unselected splenocytes or T cells with anti-Ia plus complement completely eliminated the ability of these metals to induce proliferation. In order to further delineate the subpopulation of T cells required for the metals to stimulate lymphoproliferation, experiments employing anti-T-cell monoclonal antibodies were performed. As indicated in Table 4, anti-Thy- 1.2, anti-Lyt- 1.1, and antiTABLE 4 Effect of T-Cell-Specific Monoclonal
Antibodies Plus C on Metal Stimulation cpm/culture’
Treatmentb
None
None Anti-Thy- 1.2 Anti-L+ 1 Anti-Lyt-2 Anti-L3T4
6,072 + 2,376 2,105 + 1,306 1,105 2 308* 14,325 + 3,893* 2,410+ 827
Pb 30,145 1,556 668 32,884 1,376
+ 4,683 + 162* + 125* 2 7,072 + 260*
Ni 73,928 2,548 2,411 95,543 3,176
f 3,222 31 228* -+ 991* -+ 13,614 f 1,069*
Zn 83,537 5,020 443 101,918 8,837
+ f f + +
9,333 3,330* 251* 11,393 3,953*
Note. Cells were cultured in flat-bottom 96-well plates (5 X lo5 cells/well), in M- 199 containing 5% PBS, and were pulsed and harvested on Day 6. ’ Cultures were stimulated with 100 PM metal in saline. The results represent the means + SD of triphcate cultures and are representative of two separate experiments. b Splenocytes were treated with anti-Thy-l .2 (30-H12), anti-Lyt-1.1 (CE/J anti-CBA/J alloantisera), anti-Lyt-2 (3.155), or anti-L3T4a (GKl.5) as indicated under Materials and Methods. * P < 0.05 relative to respective no-treatment control.
CATIONIC
IMMUNOMODULATION
435
L3T4 plus complement treatment was able to significantly reduce the ability of the metals to induce proliferation. Anti-Lyt-2 plus complement slightly enhanced metalinduced proliferation. Therefore, it appears that T cells bearing L3T4 and Lyt- 1 are required for the initiation of metal-induced proliferation. DISCUSSION The results reported above describe a form of immunomodulation whereby cations can modulate the immune response by activation of T cells. Evidence was presented which suggests that in vitro immunomodulation by the metals Pb, Ni, and Zn is affected by the type of basal medium employed, the presences of exogenous thiols such as 2ME, and the relative levels of cell-cell contact. Furthermore, the ability of the metals to stimulate lymphoproliferation was dependent on the presence of T cells and an Ia+ population of “accessory” cells. Previous work focused on the proliferative effects of these metals occurring in the first 3 days of culture or on the ability of these metals to alter the splenocyte proliferation induced by mitogens such as Con A and LPS on Days l-4 (2,5,6). The “early” proliferative effects have been difficult to consistently reproduce which may be due to changes in media constituents, especially with regard to the source of FBS and to low level endotoxin contamination. Low level endotoxin contamination can affect various in vitro assays and the interpretation of such assays (28). As such, this study has employed media constituents with defined levels of endotoxin contamination which are not immunomodulatory. Under these circumstances these metals do not stimulate “early” proliferation but do stimulate “late” (> Day 5) lymphoproliferation, similar to proliferation induced by soluble antigens or by allogeneic or autologous cells. The type of media employed was demonstrated to affect the ability of these metals to stimulate lymphoproliferation. In studies employing the medium M-199, Zn and Hg were able to induce human lymphocytes to proliferate late (Day 5) in culture ( 13,18). RPM1 1640, M- 199, and MEM were compared as to their abilities to support the metal-induced modulation of the primary in vitro anti-SRBC PFC response and the induction of proliferation by the metals Pb, Ni, and Zn. The media contained the same supplements, including the same amount and lot of FBS; therefore, the differences among the media must lie in some basal media constituent(s). Each medium was able to support the basal PFC and proliferative response; however, the absolute number of the PFCs induced by Pb and Ni was approximately two- to threefold greater in M-199 than in RPM1 1640 or MEM. M-199 also appeared to be the medium of choice in terms of the metal-induced proliferative response. These media differ with respect to to the amounts of GSH, PO:-, Ca*+, Fe*+, and Mg*+ with which the exogeneous metal might interact. Although RPM1 1640 is sometimes considered the medium of choice for lymphocytes (29), the basis for this is not obvious. While we did not perform an exhaustive survey of tissue culture media, it is apparent that medium selection is a critical factor in determining the immunomodulatory activity of these metals. The observed metal response may be considered weak relative to some other forms of lymphostimulation and, therefore, may be particularly sensitive to media effects as is the case with responses directed at minor histocompatibility differences (30). 2ME was shown to enhance metal-induced proliferation regardless of the basal medium employed. Indeed, no proliferation in response to Pb or Zn was
456
WARNER
AND
LAWRENCE
observed in MEM (which contains no GSH) unless 2ME was added. 2ME enhances the viability of cultured cells, is known to enhance the in vitro murine immune response (31), and has been suggested to function by minimizing random disulfide bonding of the surface of murine lymphocyte, thereby allowing the proper disulfides to form when the appropriate stimulus is received (32). These metals are capable of interacting with free sullhydryls (33) and may interact with specific cellular thiols that are involved in cellular triggering events. Cell density (cells per well) was determined to be an important factor in demonstrating metal stimulation of lymphocyte proliferation. For each of the metals tested a sharp decrease in the response was observed at densities between 1 X lo5 and 2 X lo5 cells per well in flat-bottom wells. The decrease in the response associated with decreased cell density was gradual in round-bottom wells which promote cell-cell contact, suggesting that cell-cell contact is important in the induction of proliferation by these metals. It is interesting to note that the magnitude of the response was lower in the round-bottom culture, suggesting that the extent of activation may be down regulated by excessive cell-cell interactions such as lymphocyte-macrophage interactions (34). Previous attempts to determine the nature of metal-induced immunomodulation have focused on the ability of the metal to alter responsiveness induced by T-cell or B-cell mitogens. Pb and Ni were shown to enhance the proliferative response induced by LPS which was taken to indicate that Pb and Ni acted primarily on B cells (5, 6, 20). Other investigators demonstrated that Pb alters Con A-induced proliferation while having little effect on B-cell mitogen responses (21), and as such primarily altered T-cell functions. Zn was shown to be essential for lymphocyte mitogenesis (18, 35) and was shown to enhance proliferation of spleen cells from C57Bl/6 mice stimulated with supraoptimal doses of Con A (36). None of the metals tested herein significantly affected the ability of Con A or LPS to induce lymphocyte proliferation. B cells and T cells were isolated and examined for their ability to initiate metal-induced lymphoproliferation. T-cell preparations proliferated as well as unselected splenocytes in response to metal stimulations, whereas B-cell preparations were not induced to proliferate by the metals. The possibility that the observed proliferation was due to contaminating cells does not seem likely in view of the initial purity of the respective populations and the magnitude of the response. Anti-la plus complement treatment of splenocytes completely ablated the metal-induced proliferative response. The T-cell preparations employed in these studies were not depleted of macrophages and as such contained Ia + “accessory” cells. Anti-Ia plus complement treatment of T-cell preparations also abrogated metal-induced proliferation. Clearly T cells and Ia+ accessory cells are important in metal-induced lymphoproliferation. As indicated in Table 5 a Thy-1.2+, Lyt-l+, Lyt-2-, L3T4+ cell appears to be necessary in order for the metals to stimulate proliferation. Anti-Lyt-2 plus complement treatment consistently, but not significantly, enhanced the proliferative response, suggesting that Lyt-2+ cells may be capable of slightly suppressing the response, including the background autologous response. Cations are involved in a variety of important biochemical pathways. Zn is known to be a cofactor for DNA polymerase I as well as a number of proteases (37). It has been suggested that exogeneous Zn may enhance the activity of DNA polymerase and thereby enhance [3H]thymidine incorporation (38). The fact that B cells and T cells differ in their ability to be stimulated by Zn argues against this hypothesis. Ion
CATIONIC
IMMUNOMODULATION
431
fluxes (Ca’+, Naf, K+, etc.) across the plasma membrane are thought to be intimately involved with the regulation of growth and transmembrane signaling in general (39,40). Preliminary studies in this laboratory using Ca2’ channel blockers suggest that Pb does not require active Ca2’ channels in order to modulate the in vitro PFC response (data not shown). The relative toxicity of these metals has been hypothesized to be due to their abilities to interfere with intracellular biochemical pathways which require Ca2+, especially those involving calmodulin (41). Choa et al. (42) demonstrated that Pb and Zn were able to enhance the ability of calmodulin to stimulate phosphodiesterase activity; however, Ni had little effect on calmodulin-induced phosphodiesterase activity. Studies in this laboratory demonstrated that Ni but not Pb nor Zn was able to restore the in vitro PFC in cultures chelated with EGTA (5). Ni itself is known to be a potent Ca2’ channel blocker (43). Presently, there does not appear to be a general biochemical explanation for the ability of all three metals to stimulate lymphoproliferation. Metal-induced lymphoproliferation requires the presence of both T cells and Ia+ cells, suggesting several interesting hypotheses which may account for the immunomodulatory effects of these metals on lymphoproliferation as well as the in vitro PFC response. The metals may alter the conformation of class II molecules on the surface of Ia+ cells. T cells specific for this metal-induced alteration of self would become activated in a manner analogous to the well-defined system of hapten-modified self (44), drug-modified self (45), or metal-modified self (22, 23). T cells responsive to altered self are known to exert both positive and negative effects on the in vitro PFC response (46) which could account for the observed differential effects of these metals on the in vitro PFC response. Irradiation (1000 rad) of T cells prior to culture in the presence of normal syngeneic B cells results in a significant increase in the metal (including Zn)-enhanced PFC response (G. L. Warner and D. A. Lawrence, work in progress), suggesting the presence of metal-activated radiosensitive suppressor T cells as described previously in a variety of systems (46-48). It should be noted that the ability of these metals to stimulate proliferation or modulate the in vitro PFC response is not peculiar to the CBA/J (H-23 strain of mouse since we have observed the phenomena using BALB/c (H-2d), DBA/2 (H-2”) and C57B1/6 (H-26) strains of mice (data not shown). The autologous mixed-lymphocyte response (AMLR) has recently gained much attention as a property of the normal immune response (49). As in stimulation by hapten-modified self, the autologous mixed-lymphocyte response is known to be capable of generating both helper and suppressor cells (50, 5 I), and agents which are known to enhance the expression of Ia molecules on B cells are thought to augment the AMLR (52). It is possible that the metals act directly or indirectly to enhance the expression of Ia, thereby enhancing the magnitude of the AMLR. Metallothioneins are a family of proteins rich in cysteine which are inducible by heavy metals, glucocorticoids, IFN, and IL- l(53). The metallothioneins have been thought to be primarily involved with heavy metal detoxification; however, it has recently been suggested that their primary function may be as a hydroxyl radical scavenger necessary to protect cells from oxidative insults generated by activated macrophages (54). This would suggest that IFN, IL- 1, and metallothionein are coordinately regulated. In light of the above results it would be interesting to know whether metal inducers of metallothionein are capable of inducing the expression of IFN and/or IL-l. If so, the metalinduced IFN could then induce an increase in Ia expression, thereby augmenting
438
WARNER
AND LAWRENCE
the AMLR. The possibility of metal induction of IFN could also account for the augmentation of the PFC response since (y)IFN is thought to have at least some Tcell replacing factor (TRF) activity (55). It is interesting to note that Ni and Pb enhance the SRBC-specific PFC response of T-cell-depleted splenocytes (6), a phenomenon associated with TRF activity. In conclusion, these results indicate that Pb, Ni, and Zn are capable of modulating the immune response in vitro. The possibility that these metals modulate the immune response by altering self, upsetting a normal immunohomeostatic regulatory mechanism involving T-cell recognition of self and/or affecting the production/expression of various lymphokines, is intriguing. Cation modulation of the immune response could serve as a model system for observing effects on the immune response at the cellular, biochemical, and genetic levels. REFERENCES 1. Graham, J. A., Miller, F. J., Daniels, M. J., Payne, E. A., and Gardner, D. E., Environ. Res. 16, 77, 1978. 2. Blakley, B. R., and Archer, D. L., Toxicol. Appl. Pharmacol. 61, 18, 198 I. 3. Lawrence, D. A., Infect. Zmmun. 31, 136, 1981. 4. Malave, I., and Benaim, I. R., Cell. Zmmunol. 89,322, 1984. 5. Lawrence, D. A., Toxicol. Appl. Pharmacol. 57,439, 198 1. 6. Lawrence, D. A., Znt. J. Zmmunopharmacol. 3,153, 198 1. 7. Underwood, E. J., In “Trace Elements in Human Health and Disease” (A. S. Prasad and D. Oderleas, Eds.), Vol. 2, pp. 269-280. Academic Press, New York, 1974. 8. Prasad, A. S., Nutr. Rev. 41, 197, 1983. 9. Sirover, M. A., and Loeb, L. A., Science 194, 1434, 1976. 10. Babich, H., Devanas, M. A., and Stotzky, G., Environ. Res. 37,253, 1985. 11. Cronin, E., “Contact Dermititis.” Churchill Livingstone, New York, 1980. 12. Nordlind, K., Znt. Arch. Allergy Appl. Zmmunol. 75,333, 1984. 13. Berger, N. A., and Skinner, A. M., J. Cell. Eiol. 61,45, 1974. 14. Ruhl, H., Kirchner, H., and Buchert, G., Proc. Sot. Exp. Biol. Med. 137, 1089, 197 1. 15. Nordlind, K., and Hem%, A., Znt. Arch. Allergy Appl. Zmmunol. 75,330, 1984. 16. Al-Tawil, N., Marcusson, J. A., and Moller, E., Acta Derm. Venereal. 61,5 11, 1981. 17. Nordlind, K., Znt. Arch. Allergy Appl. Zmmunol. 73, I5 1, 1984. 18. Alford, R. H., J. Zmmunol. 104,698, 1970. 19. Bendtzen, K., and Maryland, L., Scund. J. Zmmunol. 15,81, 1982. 20. Shenker, B. J., Matarazzo, W. J., Hirsch, R. L., and Gray, I., Cell. Zmmunol. 37, 19, 1977. 21. Blakley, B. R., and Archer, D. L., Toxicol. Appl. Pharmacol. 62,183,1982. 22. Pelletier, L., Pasquier, R., Hirsch, F., Sapin, C., and Druet, P., Eur. J Zmmunol. 15,460, 1985. 23. Sin&ha, F., Scheidegger, D., Garotta, G., Scheper, R., Pletscher, M., and Lanzavecchia, A., J. Zmmunol. 135,3929, 1985. 24. Lawrence, D. A,, and Weigle, W. O., J. Exp. Med. 139,943,1974. 25. Jerne, N. K., and Nordin, A. A., Science 140,405,1963. 26. Wysocki, L. J., and Sato, V. L., Proc. Natl. Acad. Sci. USA 75,2844, 1978. 27. Davidson, W. F., and Parish, C. R., J. Zmmunol. Methods. 7,29 1, 1975. 28. Eastman, A. Y., and Lawrence, D. A., Stand. J. Zmmunol. 21,35,1985. 29. Moore, G. E., Ge.mer, R. E., and Franklin, H. A., J. Amer. Med. Assoc. 199,5 19, 1967. 30. Click, R. E., Adelmann, A. M., and Azar, M. M., J. Zmmunol. 134,2948, 1985. 31. Chen, C., and Hirsch, J. G., .Z.Exp. Med. 136,604,1972. 32. Noelle, R. J., and Lawrence, D. A., Cell. Zmmunol. 60,453, 198 1. 33. Passow, H., In “Effects of Heavy Metals on Cells, Subcelhnar Elements, and Macromolecules” (J. Maniloff, L. Coleman, and M. Miller, Eds.), pp. 291-340. Thomas, Springfield, Ill., 1970. 34. Metzger, Z., Hoffeld, J. T., and Oppenheim, J. J., J. Zmmunol. 124,983,1980. 35. Zanzonico, D., Femandes, G., and Good, R. A., Cell. Zmmunol. 60,203, 198 1. 36. Malave, I., and Rondon, I., Cell. Zmmunol. 89,322, 1984.
CATIONIC 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 41. 48. 49. 50. 5 1. 52.
IMMUNOMODULATION
Parisi, A. F., and Vallee, B. L., Amer. J. Clin. Nutr. 22, 1222, 1969. Williams, R. O., and Loeb, L. A., J. Cell. Biol. 58,594, 1978. Rosoff, P. M., and Cantley, L. C., Proc. Natl. Acad. Sci. USA 80,1541, 1983. Lichtman, A. H., Segal, G. B., and Lichtman, M. A., Blood61,413,1983. Cox, J. L., and Harrison, S. D., Biochem. Biophys. Res. Commun. 115,106,1983. Chao, S. H., Sazuki, Y., Zysk, J., and Cheung, W. Y., Mol. Pharmacol. 26,15, 1984. Hagiwara, S., and Byerly, L., Annu. Rev. Neurosci. 4,69, 1981. Shearer, G. M., Lozner, E. C., Rehn, T. G., and Schmitt-Verhulst, A. M., J. Exp. Med. 141,930,1975. Nagata, N., Hurtenbach, U., and Gleichmann, E., J. Immunol. 136,136,1986. Eastman, A. Y., and Lawrence, D. A., J. Immunol. 128,926, 1982. Swain, S. L., and Dutton, R. W., J. Immunol. 118,2262, 1971. Basten, A., Miller, J. F. A. P., and Johnson, P., Transplant. Rev. 26, 130, 1975. Finnegan, A., and Hodes, R. J., J. Immunol. 136,793, 1986. Yu, D. T. Y., Chiorazzi, N., and Kunkel, H. G., Cell. Immunol. 50,305, 1980. Smith, J. B., and Knowlton, R. P., J. Immunol. 123,419, 1979. Crow, M. K., and Kunkel, H. G., Cell. Immunol. 90,555, 1985. 53. Karin, M., CeN41,9, 1985. 54. Karin, M., Imbra, R. J., Heguy, A., and Wong, G., Mol. Cell. Biol. 5,2866, 1985. 55. Brunswick, M., and Lake, P., J. Exp. Med. 161,953, 1985.
439