52, 199 –208 (1999) Copyright © 1999 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Metallothionein-Induced Suppression of Cytotoxic T Lymphocyte Function: An Important Immunoregulatory Control Jeehee Youn 1 and Michael A. Lynes 2 Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269 Received 25 March 1999; accepted 1 July 1999
Exposure to environmental toxicants can alter a variety of cellular functions that are critical to immune function. Cellular responses to these changes include increased synthesis of a number of stress proteins, some of which have been shown to have immunomodulatory capacity. One of these stress proteins, metallothionein (MT) is a low molecular weight, cysteine-rich protein that can be induced by exposure to environmental stressors as well as many inflammatory and tumorigenic agents. As a consequence, high levels of MT have been found at sites of inflammation and in certain types of neoplastic cells. In light of the suppressive effects that MT has been found to have on T-dependent humoral immunity, we investigated the potential role that MT might play in cell-mediated immune functions that could contribute to antitumor immunity. We found that MT can cause dramatic decreases in murine cytotoxic T lymphocyte (CTL) activity against allogeneic target cells. MT also reduces the proliferative response of CTLL-2 cells to cytokines, and decreases the level of major histocompatibility complex (MHC) Class I and CD8 molecules detectable on the surface of lymphocytes, while having no significant effect on the level of CD4. These findings suggest that the immunosuppressive effects of MT may at least in part reflect interference with cell– cell interactions that are ordinarily critical to cellmediated immunity. Despite this suppressive effect on CTL functioning, MT was found to augment mixed lymphocyte reactions (MLRs) in concert with increased interleukin-2 receptor (IL-2R) expression. This MT-augmented proliferation was observed in both allogeneic and syngeneic MLR. Taken together, these results indicate that MT may increase the number of immature T cells, but decrease their differentiation to the effector CTL stage. These effects of extracellular MT on T-cell function may contribute to the immunosuppression of cell-mediated immunity that has been ascribed to inducers of MT synthesis. In addition, they may point to the manipulation of MT levels as a means of reducing the undesirable immunomodulatory effects of these agents. Key Words: metallothionein; stress response protein; cell mediated immunity; cytotoxic T lymphocyte.
1
Present address: Research Institute of Immunobiology, Catholic University of Korea Medical School, 505 Banpo-dong, Seochu-ku, Seoul, 137–701, Korea. 2 To whom correspondence should be addressed at U-125, LSA 211, Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269. Fax: (860) 486-4331. E-mail:
[email protected].
Mammalian cells respond to transient environmental stress by synthesizing a specific set of stress-response proteins. Production of these proteins presumably serves to protect cells against the inimical effects of stress. However, it has been reported that stress response proteins can also participate in immune responses in a variety of ways, with both protective and non-protective consequences (reviewed in Georgopoulos and McFarland, 1993; Kaufmann, 1990). In addition to acting as molecular chaperones in the processes of immunoglobulin assembly and antigen processing, stress response proteins can serve as tumor-specific antigens (Sasaki et al., 1994; Srivastava and Maki, 1991; Srivastava and Old, 1988; Udono and Srivastava, 1993) or as targets for autoimmune attack (Holoshitz et al., 1989; Van Eden et al., 1988; Yoshino et al., 1994). Induction of certain stress proteins in tumor cells, such as GRP78 and HSP70, correlates with resistance to lysis by cytotoxic T lymphocytes (CTL), monocytes, or TNF-a (Jaattela and Wissing, 1993; Sugawara et al., 1990, 1993). Mammalian metallothionein (MT) is a cysteine-rich, stressresponse protein of low molecular weight (about 7 kDa) that binds to a number of different heavy-metal cations. Since MT gene expression can be rapidly and transiently induced by exposure to heavy metal cations (Klaassen and Lehman-McKeeman, 1989; Palmiter, 1987) or reactive oxygen species (Sato and Bremmer, 1993), it has been suggested that one function MT may serve is to protect other cellular components from these toxicants by sequestering them (Basu and Lazo, 1990; Goering and Klaassen, 1984; Hidalgo et al., 1988a; Masters et al., 1994; Sato and Bremmer, 1993; Thornally and Vasak, 1985). A variety of other cellular stressors (e.g., physical stress, phorbol esters, and bacterial endotoxin and exotoxin) have also been shown to be capable of increasing the level of MT mRNA transcripts (Choudhuri et al., 1994; De et al., 1990; Durnam et al., 1984; Garrett et al., 1992; Hidalgo et al., 1986). Following this induction, MT can be released to the extracellular environment: extracellular MT has been found in physiological fluids such as blood and urine as well as in other extracellular compartments (Bremner et al., 1987; Garvey 1984; Hidalgo et al., 1988b). The potential levels of metallothionein synthesis can be quite high: metallothionein can represent as much as 0.14% of the total protein in the
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rapidly dividing rat liver of the newborn (Gallant and Cherian, 1989), and has been found in newborn rat plasma at up to 10 ng/ml (Bremner et al., 1987). Cigarette smokers, exposed to cadmium in tobacco smoke, have also been found to have elevated serum metallothionein (Garvey, 1984). Cells placed under stressful conditions in vitro have also been reported to release metallothionein to the tissue culture medium (Thomas et al., 1987). We have previously demonstrated that extracellular MT can alter certain immune functions, which suggests that MT may be an intermediary in stress-induced immunomodulation. The finding that MT can be induced by acute-phase cytokines such as IL-1 (Karin et al., 1985; Kikuchi et al., 1993), IL-6 (Schroeder and Cousins, 1990), TNF-a (tumor necrosis factor-a) (Sato and Sasaki, 1992), and IFN-g (Freidman et al., 1984) suggests that MT may play a role in the inflammatory response. Furthermore, MT has been found to interact with the plasma membrane of immune cells, including lymphocytes and macrophages (Borghesi et al., 1996; Youn et al., 1995). This interaction may alter cell-to-cell recognition that is essential to immune functioning. Interestingly, MT interaction with lymphocytes can provoke lymphoproliferation by itself, and can also stimulate synergistic responses with either T- or B-cell mitogens (Borghesi et al.,1996; Lynes et al., 1990). Despite these mitogenic effects, MT suppresses humoral immunity both in vivo and in vitro (Lynes et al., 1993). Several lines of evidence suggest that MT can play a role in carcinogenesis. MT has been found to confer resistance to the cytotoxic effect of TNF in vitro (Leyshon-Sorland et al., 1993; Sciavolino et al., 1992). Some types of tumors express high levels of MT, including adenocarcinomas of the human thyroid (Nartey et al., 1987), human ovarian carcinomas (Bauknecht et al., 1993), and human testicular embryonal carcinoma (Kontozoglou et al., 1989) which could offer these cell types some defense against TNF. MT synthesis increases in cells found in circumstances that are acidotic, hypoxic, and nutrient-depleted, conditions that can be associated with rapid proliferation (Gallant and Cherian, 1989), and tumorigenesis. We hypothesize that MT, which can be produced during an inflammatory immune response by exposure to a variety of environmental stressors, or be produced by tumor cells, plays a role in the suppression of anti-tumor immunity that otherwise might function during oncogenesis. In this study, the behavior of CTLs, after exposure to extracellular MT, was examined. We have found that MT can contribute to dramatic decreases in CTL induction and in cytolytic activity against allogeneic target cells. In view of the principal role that CTLs play in protective immunity, changes in CTL activity following MT exposure could have crucial consequences to the effective functioning of the immune system, and could serve to explain some of the ways in which cellular stressors weaken the immune system.
MATERIALS AND METHODS Mice. BALB/cByJ and C57BL/6J mice were obtained from Jackson Laboratory, Bar Harbor, ME, or bred from animals obtained from there. Animals were housed in a room apart from other colonies, and were maintained on a 12h:12h light:dark cycle, with food and water available ad libitum. Cell lines. IL-2-dependent CTLL-2 cells (American Type Culture Collection [ATCC], TIB 214) were maintained in 60% complete RPMI 1640 containing 10% fetal bovine serum (FBS) and 40% rat spleen cell-conditioned medium. The C57BL/6 lymphoblastic cell line EL4 (ATCC, TIB 39) was cultured in complete RPMI 1640 containing 10% FBS. For all experiments, cells were incubated at 37°C in mixed gas (10% CO 2, 7% O 2, 83% N 2) in a humidified chamber (Warner and Lawrence, 1988). Media and reagents. Rabbit liver Zn,Cd-metallothionein (a mixture of MT-I and MT-II), obtained from Sigma Chemical Co. (St. Louis, MO), was reconstituted in phosphate buffered saline (PBS), pH 7.2, and used for all experiments at 20 mM final concentration after 0.2 m filter sterilization. This metallothionein has been found to have 4 to 5 available thiols, as detected by their reaction with CPM (N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide; Molecular Probes, Eugene, OR), suggesting that significant oxidation has not occurred. Moreover, measurements of the associated metals (2 Zn and 5 Cd per MT) also indicate that the protein is in a non-oxidized state. The use of a mixture of metallothionein-I and -II was chosen to represent what would be synthesized after exposure to a heavy metal stressor. RPMI 1640 (GIBCO/BRL, Gaithersburg, MD) was supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), sodium bicarbonate, 0.1 mM non-essential amino acids, 0.1 mM sodium pyruvate, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin (GIBCO/BRL), and 1X Basal Medium Eagle vitamin solution (Sigma). For CTL induction, 10% heat inactivated FBS (GIBCO/BRL) was used. For lactic acid dehydrogenase (LDH) release assays, M199 without phenol red (GIBCO/BRL) was supplemented with sodium bicarbonate, 25 mM HEPES buffer (GIBCO/BRL), 50 mg/ml gentamycin (Sigma), and 2% bovine serum albumin (Sigma). Rat spleen cell-conditioned medium as a source of IL-2 was generated as described previously (Fitch and Gajewski, 1991). In brief, spleen cells of Hsd: Sprague-Dawley rats (Harlan-Sprague-Dawley, Inc., Indianapolis, IN) were incubated at 10 6 cells/ml in RPMI 1640 medium containing 5% FBS plus 5 mg/ml Con A in 5% CO 2 in humidified air. After 40 to 44 h of incubation, supernatant was harvested by centrifugation at 200 3 g for 10 min, and then at 800 3 g for 10 min. Supernatant was used for the culture of CTLL-2 cells after filter sterilization through a 0.2 m filter. Antibodies. Anti-mouse CD4-FITC (fluorescein isothiocyanate) (clone GK 1.5), anti-mouse CD8a-FITC (clone 53– 6.7), and goat anti-rat IgG (whole molecule)-FITC were obtained from the Flow cytometry facility of the Jackson Laboratory (Bar Harbor, ME), PharMingen (San Diego, CA), and Sigma, respectively. 7D4 hybridoma cells (ATCC, CRL 1698) which produce antimouse IL-2R monoclonal antibody were grown as ascites in BALB/cByJSmnscid/J mice and antibody was prepared from ascites fluid by ammonium sulfate precipitation. Anti-H-2K b monoclonal antibody was purified from spent medium of HB158 cell (ATCC) culture using a protein A-agarose column (Schleicher and Schuell, Inc., Keene, NH). Goat anti-mouse IgG (whole molecule)-FITC was purchased from Sigma. Induction and measurement of CTL activity. CTLs were generated by culture of BALB/cByJ splenic T cells with allogeneic stimulator cells. T cells were isolated from spleen cells by nylon wool non-adherence, as described by Hathcock (1991). Erythrocyte-depleted C57BL/6J spleen cells were used as allogeneic stimulators after treatment with mitomycin C (Sigma) at 25 mM/ml final concentration for 20 min at 37°C in the dark, followed by 3 washes with unsupplemented RPMI 1640. Mixtures of 8 3 10 6 responder T cells and 4 3 10 6 stimulator spleen cells were incubated together in 4 ml of complete RPMI 1640 containing 10% heat inactivated FBS in 60 3 15 mm petri dishes (Beckton Dickinson, Mountain View, CA) at 37°C in mixed gas for 5 days. CTL activity from these cultures was measured by specific lactic acid dehy-
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METALLOTHIONEIN MODIFIES T-CELL FUNCTIONS drogenase (LDH) release, according to a previously described method (Korzeniewski and Callewaert, 1983). In brief, effector cells recovered after allogeneic stimulation were washed 3 times in M199 supplemented with 2% bovine serum albumin. Wells of U-bottom 96-well microtiter plates (Corning Glass Works, Corning, NY) received 10 4 EL4 target cells in 100 ml of supplemented M199, plus effector cells in numbers as described in the results section, for a total volume of 200 ml/well. The microtiter plates were centrifuged for 4 min at 150 3 g and incubated at 37°C in mixed gas for 4 h. After adding 50 ml of cold M199-albumin to each well and centrifuging for 10 min at 300 3 g, 100 ml aliquots of supernatant were transferred to the corresponding wells of a second microtiter plate (Immulon 2 ELISA plate, Dynatech Laboratories, Inc., Alexandria, VA). One hundred ml of LDH substrate mixture (54 mM L-lactate, 0.66 mM 2-p-iodophenyl-3-p-nitrophenyl tetrazolium chloride, 0.28 mM phenazine methosulfate, and 1.3 mM nicotinamide adenine dinucleotide in 0.2 M Tris buffer, pH 8.2) was added to each well. Kinetic color development was determined in a Tmax ELISA microplate reader (Molecular Devices, Menlo Park, CA) at dual wavelengths of 490 nm and 750 nm. The percent cytotoxicity was calculated as follows: % C 5 [(E – S)/(M – S)] 3 100, where E represents the LDH activity in experimental samples; S, the spontaneous release of LDH activity from target cells incubated in the absence of effector cells; and M, the maximal release of LDH activity after 4 cycles of freezing and thawing. In experiments designed to determine the action of MT, it was added at different points in the CTL assay. Responder T cells and allogeneic stimulator splenocytes were co-incubated in the presence of 20 mM MT to examine the effect of MT on effective CTL generation. After 5 days of incubation, cells were washed 3 times with M199-albumin, followed by a CTL effector assay. In order to assess if MT alters the capacity of stimulator cells to activate precursor CTLs, C57BL/6J spleen cells were preincubated with 20 mM MT for 20 h and washed to remove unbound MT prior to co-culture with responder cells. In another set of experiments, EL4 cells were preincubated with 20 mM MT for 20 h, washed, and used as target cells for demonstration of MT effect on the target cell’s capacity to be recognized by CTLs. Mixed lymphocyte reaction (MLR). MLR cultures were carried out in parallel with CTL cultures in separate 96-well microtiter plates. BALB/cByJ T lymphocytes, purified by nylon-wool non-adherence, were prepared in supplemented RPMI 1640 containing 10% heat-inactivated FBS at 4 3 10 6 cells/ml and 100 ml of this cell suspension was added to individual wells to serve as responder T cells. Mitomycin C-treated allogeneic C57BL/6J or syngeneic BALB/cByJ splenocytes (2 3 10 6/ml) were added in 100-ml aliquots to each well of the plates containing responder cells. These mixtures of cells were incubated for 5 days, further incubated with 3H-thymidine (0.5 mCi/well, Amersham, Arlington Heights, IL) for 6 h, and then harvested for determination of thymidine incorporation. Fluorescence labeling. Eight 3 10 6 splenic T cells of BALB/cByJ and 4 3 10 6 mitomycin C-treated spleen cells of C57BL/6J were incubated together in the presence or absence of 20 mM MT in 4 ml of complete RPMI 1640 containing 10% heat inactivated FBS, for 5 days. After incubation, cells were washed with M199, resuspended in M199 containing 2% goat serum and incubated with anti-CD4-FITC, anti-CD8-FITC or rat anti-mouse IL-2R (7D4) on ice for 30 min. Cells incubated with anti-IL-2R antibody were washed with M199 3 times and incubated with goat anti-rat IgG (whole molecule)-FITC (Sigma) on ice for another 30 min. Cells were again washed 3 times, and fluorescence intensity was analyzed using a FACScan flow cytometer (Beckton Dickinson, Mountain View, CA). Propidium iodide staining was used to exclude dead cells from analysis. To assess the effect of MT on MHC class I expression, C57BL/6J thymocytes were incubated in complete RPMI containing 10% FBS in the presence of 20 mM MT or PBS for 2 days. After incubation, cells were harvested and washed 3 times in M199. Cells were resuspended in M199 plus 2% goat serum and incubated with anti-H-2K b monoclonal antibody on ice for 30 min, followed by washing in M199. Cells were then incubated with goat anti-mouse IgG-FITC (Sigma) on ice for another 30 min, washed 3 times in M199, stained
with propidium iodide, and analyzed by epi-illumination using a fluorescent microscope. Proliferation assay of CTLL-2. CTLL-2 cells were cultured in a mixture of 60% complete RPMI and 40% rat spleen cell-conditioned medium used as a source of CTLL-2 cell growth factors (Gillis and Smith, 1977). Cells were extensively washed in unsupplemented RPMI 1640 medium and resuspended in the mixture of complete RPMI plus rat-conditioned medium at various ratios. Five 3 10 3 CTLL-2 cells were incubated in the presence or absence of 20 mM MT in a 96-well plate. After 20 h of incubation, 25 ml of 3H-thymidine (0.5 mCi/well) was added to each well. The cultures were harvested 6 h later with a multiple automatic harvester (Cambridge Instruments, Watertown, MA) and incorporated 3H-thymidine was measured. To address the possibility of MT interaction with component(s) of the conditioned medium, 20 mM MT was preincubated with various concentrations of the conditioned medium for 24 h. This preincubated, rat-conditioned medium was added to CTLL-2 cell cultures at 0, 20, or 40% final concentrations. After a 20-h incubation, 3H-thymidine incorporation was determined as described above.
RESULTS
Metallothionein Reduces the Activity of Allospecific Cytotoxic T Lymphocytes The ability of MT to alter the course of CTL interactions with target cells was examined by adding MT during several individual stages of the CTL assay. First, MT was added to cultures in which CTL cells differentiate. BALB/cByJ (H-2 d) T cells were co-cultured with mitomycin C-treated C57BL/6J (H-2 b) splenocyte stimulators in the presence of 20 mM MT. After this 5-day culture, the cells were washed and tested for CTL function against EL4 (H-2 b) target cells. MT in these experiments was found to reduce specific CTL activity by more than 70% at the 30:1 and 10:1 E:T ratios (Fig. 1A). The possibility that MT may affect the capacity of stimulator cells to be recognized by allogeneic T cells was also examined. Mitomycin C-treated C57BL/6J splenocytes were preincubated with 20 mM MT for 20 h, washed to remove free MT, and then co-incubated with BALB/cByJ T cells for 5 days. The effectors recovered from these cultures displayed a significant decrease in cytotoxic activity against EL4 target cells at the highest E:T ratio: there was a 26% reduction of CTL activity when MT was present in the preculture of the stimulator cells (Fig. 1B). The influence of MT on CTL recognition of target cells was examined by pretreatment of EL4 target cells with MT before incubation of these cells with effector cells. Target cells were preincubated with 20 mM MT for 20 h, washed, and then used in the CTL effector assay. Exposure to MT in this way also caused a significant decrease in target cell killing (approximately 25 % of control at the 30:1 E:T ratio) (Fig. 1C). In contrast, MT added during effector-target cell co-incubation period for 4 h did not alter cytotoxicity (data not shown). In all of these experiments, there was no alteration in effectorcell viability in cultures to which MT was added (data not shown). Taken together, these results suggest that metallothionein interferes with the differentiation of CTLs during a 5-day co-culture with target. This interference appears to relate to the
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FIG. 2. MT effects on allogeneic mixed-lymphocyte reaction. Splenic T lymphocytes of BALB/cByJ mice (R) and mitomycin C-treated splenocytes of C57BL/6J mice (S) were co-cultured in the presence of MT or its vehicle control (PBS). After 5 days of culture, 3H-thymidine incorporation was assessed as a measure of proliferation. Each value represents the average of triplicates. The standard deviation was less than 10% of each average value.
initial interaction with target, since preincubation with metallothionein of either effector cells or target cells decreases the interaction, while metallothionein present during the lytic effector phase has no effect. We did note a metallothioneinmediated increase in cellularity in the CTL assays, and proceeded to assess this effect of metallothionein exposure in the MLR assays described in the next section. Metallothionein Alters Cellular Responses in the Mixed Lymphocyte Reaction
FIG. 1. MT effects on cytotoxic T lymphocyte function. (A) Splenic T lymphocytes of BALB/cByJ mice were co-cultured with mitomycin C-treated splenocytes of C57BL/6J in the presence ( z z z ) or absence (—) of 20 mM MT for 5 days. Effector cells recovered from this mixed culture were assayed for CTL activity as measured by lactic acid dehydrogenase released from lysed EL4 target cells. (B) Splenocytes of C57BL/6J mice were preincubated with 20 mM MT ( z z z ) or PBS (—) for 20 h and then unbound MT was washed away. These stimulator cells co-cultured with splenic T cells of BALB/cByJ mice for 5 days for CTL induction. Effector cells were assayed for cytolytic activity against EL4 target cells. (C) Splenic T lymphocytes of BALB/cByJ mice were co-cultured with mitomycin C-treated splenocytes of C57BL/6J for 5 days.
In light of the effects on CTL differentiation, we next examined the contributions MT might make to recognition of allogeneic targets in proliferation assays. The presence of the stress protein MT in mixed lymphocyte reactions (MLR) augmented proliferation induced by alloantigen. T lymphocytes from BALB/cByJ (H-2 d) cultured with mitomycin C-treated splenocytes from C57BL/6J (H-2 b) and 20 mM MT showed 4to 12-fold increases in 3H-thymidine incorporation as compared to vehicle-treated controls (Fig. 2). Addition of MT to cultures containing only H-2 d responder T lymphocytes or mitomycin C-treated H-2 b stimulators did not evoke proliferation, indicating that the effect MT has is dependent upon other changes that must take place in the MLR culture. Although MT did not directly induce proliferation of H-2 d T lymphocytes, it is possible that this stress protein could serve
Effector cells recovered from this mixed culture were incubated with EL4 target cells which had preincubated with either 20 mM MT ( z z z ) or PBS ( —) for 20 h. Cytotoxicity was calculated as described in Materials and Methods. Results are reported as the average of triplicates 6 sd and are representative of at least 3 independent experiments. Results that are significantly different from control (PBS-treated) are marked with an asterisk: * p , 0.02 (t-test).
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FIG. 3. The effect of MT-pretreated stimulation on allogeneic MLR. Mitomycin C-treated splenocytes (stimulators) from C57BL/6J mice were preincubated with 20 mM MT or PBS for 20 h, washed with medium, and added to T lymphocytes from BALB/cByJ mice (responders) (A). Responders were similarly preincubated with MT (B) before co-culture with stimulators. After 5 days, cells were pulsed with 3H-thymidine and assessed for proliferation. Panel C shows the effect of the continuous presence of MT during the responder/stimulator co-culture. Each panel depicts the average thymidine incorporation of 2 to 5 independent experiments (each experiment is represented by a unique symbol). The stimulation index (SI) represents cpm of the culture after addition of MT divided by that with vehicle alone.
as a co-stimulator to augment responses to alloantigen. This possibility was examined by preincubating each individual cell population with MT prior to the mixed-cell co-culture. H-2 b stimulator or H-2 d responder cells were precultured for 20 h, washed, and then combined in the MLR culture. Pretreatment of stimulator lymphocytes with MT-augmented allospecific responses by 1.5 to 2.5-fold (Fig. 3A), while pretreatment of the responder cells increased proliferation by 1.5 to 1.7-fold (Fig. 3B). By comparison, cultures in which MT was added directly to the 5-day MLR had 4- to12-fold increases in alloantigen-driven proliferation (Fig. 3C). Finally, we examined the ability of MT to alter the course of an autologous MLR. Syngeneic responder and stimulator cells, continuously co-cultured in the presence of MT, showed a modest increase in the responder T-cell proliferation when compared to controls (Fig. 4C). These modest increases were even smaller when MT was preincubated with either stimulators or responders before the MLR co-culture period (Figs. 4A and 4B).
(Fig. 5B). CD4 levels were unaffected by the presence of MT in these cultures (data not shown). The declines in CD8 1 cells correlate with declines in Thy-1 1 T cells in splenocyte cultures that have been incubated with 20 mM metallothionein. After culturing for 2 days in vehicle control, splenocyte cultures had 38.1% Thy-1 1 cells, while the culture with metallothionein had only 14.6% Thy-1 1 cells MT was also responsible for decreased MHC class I antigen expression. Thymocytes cultured with or without 20 mM MT were labeled with anti-H-2K b antibody and examined under an epi-illumination fluorescent microscope. Dead cells were excluded from the analysis by propidium iodide staining. Incubation of cells with MT reduced the number of anti-H-2K blabeled cells visible under the microscope from 40.3 6 7.5% to 18.8 6 4.5%.
MT Addition in Allogeneic MLR Changes the Pattern of Cell Surface Marker Expression
IL-2 dependent CTLL-2 cells were cultured in Con A-stimulated rat spleen cell-conditioned medium as a source of exogenous cytokines. MT added directly to these cultures containing conditioned medium did not alter CTLL-2 proliferation as compared to control cultures (Fig. 6A). However, exogenous MT preincubated with conditioned medium for 2 days at 37°C before addition of this supplement to CTLL-2 cultures was responsible for significant decreases in CTLL-2 proliferation (Fig. 6B). MT did not alter cell viability in any of these cultures (data not shown).
In order to assess the changes in cell-surface marker expression that could occur during the MLR, cells were harvested after culture with MT and analyzed by flow cytometry. The number of IL-2R 1 cells was substantially increased by coculture with MT (Fig. 5A). After 5 days of culture the number of lymphocytes expressing IL-2R increased from 14% in control cultures to 39% in MT treated cultures. In contrast, CD8 levels decreased on cells co-cultured in the presence of MT
Metallothionein Alters the Responsiveness of CTLL-2 Cells to Conditioned Medium
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FIG. 4. MT augments proliferation in an autologous MLR co-culture system. Erythrocyte-depleted BALB/cJ splenocytes were treated with mitomycin C for use as stimulators, and syngeneic splenic T cells were used as responders. In one series of cultures (no preculture) these cells were co-cultured for 5 days in the presence or absence of MT (C). In the second series of cultures, the stimulator cells (A) or responder cells (B) were precultured for 24 h in the presence of absence of MT, washed, and then co-cultured with syngeneic responder or stimulator cells, respectively, in complete medium without added MT. At the end of the 5-day culture, cell proliferation was assessed by 3H-thymidine incorporation. Results are reported as the average of triplicates and each symbol represents one of the independent experiments.
DISCUSSION
The experiments described here were designed to investigate the immunomodulatory effect of MT in the context of CTL function. In this report, we demonstrate that MT interferes with specific CTL activity. MT, added during the differentiation phase of a CTL assay, exerted the most dramatic suppressive
effect, while MT preincubated with target cells evoked results that are more modest. Metallothionein present during the effector phase of the CTL assay had no discernable impact on target-cell lysis. These results suggest that metallothionein can interfere with some phase of the T-cell interaction with target cells. In contrast to this effect on CTL function, MT increased
FIG. 5. Fluorescence labeling of cell-surface markers in allogeneic MLR. Mixed-lymphocytes culture of BALB/cByJ T cells and mitomycin C treated splenocytes from C57BL/6J in the presence of MT ( z z z ) or PBS as vehicle control (—) for 5 days were labeled with rat anti-IL-2R plus goat anti-rat IgG (whole molecules)-FITC (A), or anti-CD8-FITC (B). Fluorescence intensity was examined by FACScan flow cytometry. Propidium iodide staining was used to gate for live cells.
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FIG. 6. MT effects on the CTLL-2 response to conditioned medium. CTLL-2 cells were cultured in 0, 20, or 40% rat-conditioned medium in the presence or absence of MT (A), or in 0, 20, or 40% rat-conditioned medium that had been preincubated with MT or PBS for 2 days (B). After CTLL-2 incubation with the conditioned medium for 20 h, the cells were pulsed with 3 H-thymidine and proliferation assays were performed. Each column represents the average cpm of triplicate 6 SD. This result is representative of 3 independent experiments. Results that are significantly different from control (PBS-treated) are marked with an asterisk: *p , 0.02 (t-test).
the levels of detectable IL-2R, and enhanced MLR responses. This latter effect was observed both in allogeneic and syngeneic MLR combinations of responders and stimulators. The effect of the suppression of CTL function and enhanced cell proliferation in an in vivo situation may be to reduce the protective anti-tumor response while simultaneously augmenting tumor cell proliferation, and might represent a mechanism by which tumor cells can evade effective anti-tumor responses. One way in which MT might alter CTL function is by interference with the plasma membrane-associated molecules that are essential to CTL recognition of target cells. Our results show that metallothionein reduced detectable MHC class I and CD8: each of these surface molecules is essential to the successful interaction between CTL and target cell. MHC class I expression is often reduced in tumor cells, where it has been shown that tumor progression in vivo can be inversely proportional to the level of MHC class I to be found at the cell surface (Bernards et al., 1983; Ferrone and Marincola, 1995; Sanderson and Beverly, 1983; Wallich et al., 1985). CD8 coreceptor molecules have also been demonstrated to play an important role in CTL functioning (Al-Ramadi et al., 1995; Crooks and
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Littman, 1994), and declines in CD8 availability may also interfere with CTL effectiveness. There are several different ways in which metallothionein might alter the expression of cell-surface markers. First, metallothionein might interfere with the availability of these antigens by forming mixed disulfides with available cysteines, within the structure of expressed molecules. The human MHC protein, HLA-B27, has such a free thiol in the antigen binding cleft that is susceptible to chemical modifications such as oxidation (Whelan and Archer, 1993) and could be an example of such a target. These authors suggest that the disease associations with HLA-B27 may derive from its unstable nature and the potential for chemical modification or adducts at this site. MT’s chemical interactions with surface proteins might interfere with their ability to function as receptors or indirectly interfere with their accessibility to other surface molecules. Our previous results have shown that metallothionein can bind to the surface of lymphocytes, and that this binding can be modified by 2-mercaptoethanol (Borghesi et al., 1996). T-lymphocyte functions have been shown to be very sensitive to changes in membrane associated thiol chemistry (Noelle and Lawrence, 1981). Alternatively, membrane binding by metallothionein might be more specific (as has been shown for metallothionein binding to specific receptors on astrocytes (El Refaey et al., 1997) and may initiate changes in some lymphocyte signal transduction cascade via a traditional ligand/receptor interaction. Metallothionein interactions with cells may also serve to shift the developmental program of the cell, acting via an interaction with NF-kB, a transcription factor with multiple roles to play in immune function (Wu et al, 1996). Recent studies have suggested that metallothionein may interact directly with the p50/RelA heterodimer, stabilizing its interaction with DNA (Van Antwerp, 1996). If so, metallothionein synthesized as a consequence of metallothionein exposure (McKim et al, 1992) may alter the developmental program of the metallothionein-exposed cell. Another intriguing possibility is that the declines in CTLmediated killing that occur in the presence of metallothionein may result from metallothionein’s ability to render cells resistant to the induction of apoptosis. Metallothionein confers resistance to TNF-induced apoptosis (Leyshon-Sorland et al., 1993; Sciavilino et al, 1992), and antisense inhibition of metallothionein increases apoptosis (Tsangaris and TzortzatouStathopoulou, 1998). Metallothionein-null cells exhibit increased apoptosis (Kondo et al., 1997; Abdel-Mageed and Agrawal, 1997). Intriguingly, extracellular metallothionein can also initiate apoptosis in some circumstances (Ishido et al., 1999), demonstrating that the integrated role of metallothionein in the regulation of apoptosis in vivo is likely to be complex. Metallothionein may also act to shift gene function by changing the amount of zinc available to zinc-finger transcription factors such as Sp-1 (Remondelli et al., 1997). The con-
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sequence of these changes might be related to the decreases in MHC class I, and CD8 expression that we observed, as well as to the declines in CTL function. In addition to the suppression of CTL function, we observed that MT caused a decrease in the proliferation of CTLL-2 cells, an IL-2-dependent cytotoxic T-lymphoma line, when MT was preincubated with the conditioned medium which served as a source of this cytokine (Fitch and Gajewski, 1991). This finding suggests that MT can interact with soluble cytokine(s) and can result in the inhibition of signal transduction triggered by occupancy of the relevant receptor(s). Two free thiols associated in the IL-2 polypeptide are potential targets for MT binding. In contrast to the suppressive effect of MT on CTL activation, BALB/cByJ T cells stimulated with C57BL/6J splenocytes in the presence of MT proliferate more than those cultured in the absence of MT. This proliferative effect on primary culture lymphocytes has also been reported in other assay systems. In a previous report, MT was found to elicit proliferation of unfractionated splenocytes, both alone and synergistically, when added in the context of polyclonal activators (Lynes et al., 1990). This mitogenic capacity depends upon the available thiols in the MT molecule (Borghesi et al., 1996). T cells co-cultured with syngeneic or allogeneic stimulator cells that had been preincubated with MT also exhibit an increase in proliferation, suggesting that MT bound to stimulator cell surface might augment costimulatory capacity of stimulator cells. Thus, these results show that while MT increased the number of responder cells in CTL culture, functioning of these cells was reduced. One interpretation of these apparently conflicting observations is that MT may augment lymphocyte proliferation while interfering with events important for functional maturation. Thus, MT may promote an increased number of immature CTLs but prevent differentiation to the highly effective CTLs. We have observed similar effects of MT on the humoral immune response (Lynes et al., 1993). In those experiments, MT enhanced B cell proliferation but reduced the capacity of these cells to produce antigen-specific antibodies. In concert with the increased proliferation of responder cells that occurs in the presence of MT, an increase in the proportion of IL-2R1 cells was observed. Another protein with free thiols has been reported to have similar effects on IL-2R expression. Adult T cell leukemia-derived factor (ADF), initially described as an IL-2R a chain inducer, exhibits a synergistic activity on the mitogen- or cytokine-induced proliferation of virally transformed lymphoid cell lines (Tagaya et al., 1988; Yamauchi et al., 1992). ADF has an active dithiol group (-Cys-Gly-ProCys-) and acts as a thiol-dependent reducing protein. Interestingly, mammalian MT protein contains the conserved Cys-XX-Cys sequences (X represents an amino acid residue other than cysteine). Both MT and ADF are members of a class of secretory proteins that does not possess hydrophobic signal
sequences and appears to leave the cell by a secretory pathway distinct from the classical route (Muesch et al., 1990). Taken together, these results demonstrate that MT may alter several mechanisms that combine to interfere with effective cellular immune responses. In the context of the crucial role that CTLs play in anti-tumor immunity, overexpression of MT by rapidly dividing tumor cells, by bystander cells exposed to the anoxic environment of the tumor, or by cells responding to acute phase cytokines, metallothionein may allow tumor cells to evade the host defense immune response. Moreover, genetic disparities in the MT responses of individuals (Yurkow and Makhijani, 1998) may contribute to the relative susceptibilities of these individuals to MT-mediated alterations in tumorigenesis. ACKNOWLEDGMENTS The authors would like to acknowledge the valuable contribution of Dr. Lisa Borghesi for her critical review of the manuscript. The authors would also like to acknowledge the skilled assistance of the animal-care technicians Marie Joyner and Danette Myers. This work was supported in part by NIEHS grant ES07408.
REFERENCES Abdel-Mageed, A., and Agrawal, K. C. (1997). Antisense down-regulation of metallothionein induces growth arrest and apoptosis in human breast carcinoma cells. Cancer Gene Ther. 4, 199 –207. al-Ramadi, B. K., Jelonek, M. T., Boyd, L. F., Margulies, D. H., and Bothwell, A. L. M. (1995). Lack of strict correlation of functional sensitization with the apparent affinity of MHC/peptide complexes for the TCR. J. Immunol. 155, 662– 673. Basu, A., and Lazo, J. S. (1990). A hypothesis regarding the protective role of metallothioneins against the toxicity of DNA interactive anticancer drug. Toxicol. Lett. 50, 123–135. Bauknecht, T., Angel, P., Kohler, M., Kommoss, F., Birmelin, G., Pfleiderer, A., and Wagner, E. (1993). Gene structure and expression analysis of the epidermal growth factor receptor, transforming growth factor-alpha, myc, jun, and metallothionein in human ovarian carcinomas. Cancer 71, 419 – 429. Bernards, R., Schrier, P. I., Houweling, A., Bos, J. L., van der Eb, A. J., Zijlstra, M., and Melief, C. J. M. (1983). Tumorigenicity of cells transformed by adenovirus type 12 by evasion of T-cell immunity. Nature 305, 776 –779. Borghesi, L. A., Youn, J., Olson, E. A., and Lynes, M. A., (1996). Interactions of metallothionein with murine lymphocytes: Plasma membrane binding and proliferation. Toxicology 108, 129 –140. Bremner, I., Mehra, R. K., and Sato, M. (1987). Metallothionein in blood, bile and urine. EXS 52, 507–517. Choudhuri, S., McKim, J. M., Jr., and Klaassen, C. D. (1994). Induction of metallothionein by superantigen bacterial exotoxin: Probable involvement of the immune system. Biochim. Biophys. Acta 1225, 171–179. Crooks, M. E., and Littman, D. R. (1994). Disruption of T lymphocyte positive and negative selection in mice lacking the CD8 beta chain. Immunity 1, 277–285. De, S. K., McMaster, M. T., and Andrews, G. K. (1990). Endotoxin induction of murine metallothionein gene expression. J. Biol. Chem. 265, 15267– 15274. Durnam, D. M., Hoffman, J. S., Quaife, C. J., Benditt, E. P., Chen, H. Y.,
METALLOTHIONEIN MODIFIES T-CELL FUNCTIONS Brinster, R. L., and Palmiter, R. D. (1984). Induction of mouse metallothionein-I mRNA by bacterial endotoxin is independent of metals and glucocorticoid hormones. Proc. Natl. Acad. Sci. U S A 81, 1053–1056. El Rafaey, H., Ebadi, M., Kuszynski, C. A., Sweeney, J., Hamada, H. M., and Hamed, A. (1997). Identification of metallothionein receptors in human astrocytes. Neurosci. Lett. 231, 131–134. Ferrone, S., and Marincola, F. M. (1995). Loss of HLA class I antigens by melanoma cells: Molecular mechanisms, functional significance, and clinical relevance. Immunol. Today 16, 487– 494. Fitch, F. W., and Gajewski, T. F. (1991). Production of T cell clones. In Current Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, Eds.), pp. 3.13.1–3.13.11. Greene Publishing and Wiley-Interscience, New York. Friedman, R. L., Manly, S. P., McMahon, M., Kerr, I. M., and Stark, G. R. (1984). Transcriptional and post-transcriptional regulation of interferoninduced gene expression in human cells. Cell 38, 745–755. Gallant, K. R., and Cherian, M. G. (1989). Metabolic changes in glutathione and metallothionein in newborn rat liver. J. Pharmacol. Exp. Ther. 249, 631– 637. Garrett, S. H., Xiong, X., Arizono, K., and Brady, F. (1992). Phorbol ester induction of rat hepatic metallothionein in vivo and in vitro. Int. J. Biochem. 24, 1669 –1676. Garvey, J. S. (1984). Metallothionein: Structure/antigenicity and detection/ quantitation in normal physiological fluids. Environ. Health Prospect. 54, 117–127. Georgopoulos, C., and McFarland, H. (1993). Heat-shock proteins in multiple sclerosis and other autoimmune diseases. Immunol. Today 14, 373–375. Gillis, S. and Smith, K. (1977) Long-term culture of tumour-specific cytotoxic T cells. Nature 268, 154 –156. Goering, P. L., and Klaassen, C. D. (1984). Tolerance to cadmium-induced hepatotoxicity following cadmium pretreatment. Toxicol. Appl. Pharmacol. 74, 308 –313. Hathcock, K. S. (1991). T cell enrichment by nonadherence to nylon. In Current Protocols in Immunology, (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, Eds.), pp. 3.2.1–3.2.4. Greene Publishing and Wiley-Interscience, New York. Hidalgo, J., Armario, A., Flos, R., Dingman, A., and Garvey, J. S. (1986). The influence of restraint stress in rats on metallothionein production and corticosterone and glucagon secretion. Life Sci. 39, 611– 616. Hidalgo, J., Campmany, L., Borras, M., Garvey, J. S., and Armario, A. (1988a). Metallothionein response to stress in rats: Role in free-radical scavenging. Am. J. Physiol. 255, E518 –524. Hidalgo, J., Giralt, M., Garvey, J. S., and Armario, A. (1988b). Physiological role of glucocorticoids on rat serum and liver metallothionein basal and stress conditions. Am. J. Physiol. 254, E71–78. Holoshitz, J., Koning, F., Coligan, J. E., de Bruyn, J., and Strober, S. (1989). Isolation of CD4-CD8: Mycobacteria-reactive T lymphocyte clones from rheumatoid arthritis synovial fluid. Nature 339, 226 –229. Ishido, M., Tohyama, C., and Suzuki, T. (1999). Cadmium-bound metallothionein induces apoptosis in rat kidneys, but not in cultured kidney LLC-PK1 cells. Life Sci. 64, 797– 804. Jaattela, M., and Wissing, D. (1993). Heat shock proteins protect cells from monocyte cytotoxicity: Possible mechanism of self-protection. J. Exp. Med. 177, 231–236. Karin, M., Imbra, R. J., Heguy, A., and Wong, G. (1985). Interleukin 1 regulates human metallothionein gene expression. Mol. Cell. Biol. 5, 2866 – 2869. Kaufmann, S. H. E. (1990). Heat shock proteins and the immune response. Immunol. Today 11, 129 –136. Kikuchi, Y., Irie, M., and Kasahara, T., Sawada, J., and Terao, T. (1993).
207
Induction of metallothionein in a human astrocytoma cell line by interleukin-1 and heavy metals. FEBS Lett. 317, 22–26. Klaassen, C. D., and Lehman-McKeeman, L. D. (1989). Induction of metallothionein. J. Am. Coll. Toxicol. 8, 1315–1320. Kondo, Y., Rusnak, J. M., Hoyt, D. G.. Settineri, C. E., Pitt, B. R., and Lazo, J. S. (1997). Enhanced apoptosis in metallothionein null cells. Mol. Pharmacol. 52, 195–201. Kontozoglou, T. E., Banerjee, D., and Cherian, M. G. (1989). Histochemical localization of metallothionein human testicular embryonal carcinoma cells. Virchows Arch. A: Patho. Anat. Histopathol. 415, 545–549. Korzeniewski, C., and Callewaert, M. (1983). An enzyme-release assay for natural cytotoxicity. J. Immunol. Methods 64, 313–320. Leyshon-Sorland, K., Morkrid, L., and Rugstad, H. (1993). Metallothionein: A protein conferring resistance in vitro to tumor necrosis factor. Cancer Res. 53, 4874 – 4880. Lynes, M. A., Borghesi, L. A., Youn, J., and Olson, E. A. (1993). Immunomodulatory activities of extracellular metallothionein: I. Metallothionein effects on antibody production. Toxicology 85, 161–177. Lynes, M. A., Garvey, J. S., and Lawrence, D. A. (1990). Extracellular metallothionein effects on lymphocyte activities. Mol. Immunol. 27, 211– 219. Masters, B. A., Kelly, E. J., Quaife, C. J., and Brinster, R. L. (1994). Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. U S A 91, 584 –588. McKim, J. M., Liu, J., Liu, Y. P., and Klaassen, C.D. (1992). Induction of metallothionein by cadmium-metallothionein in rat liver: A proposed mechanism. Toxicol. Appl. Pharmacol. 112, 318 –323. Muesch, A., Hartmann, E., Rohde, K., Rubartelli, A., Sitia, R., and Rapoport, T. A. (1990). A novel pathway for secretory proteins? Trends in Biochem. Sci. 15, 86 – 88. Nartey, N., Cherian, M. G., and Banerjee, D. I. (1987). Immunohistochemical localization of metallothionein in human thyroid tumors. Am. J. Pathol. 129, 177–182. Noelle, R. J., and Lawrence, D. A. (1981). Modulation of T-cell function: II. Chemical basis for the involvement of cell-surface thiol-reactive sites in control of T-cell proliferation. Cell. Immunol. 60, 453– 469. Palmiter, R. (1987). Molecular biology of metallothionein gene expression. EXS 52, 63– 80. Remondelli P, Moltedo, O, and Leone, A (1997). Regulation of ZiRF1 and basal SP1 transcription factor MRE-binding activity by transition metals. FEBS Lett. 416, 254 –258 Sanderson, A. R., and Beverly, P. C. L. (1983). Interferon, b-2-microglobulin and immunoselection: In the pathway to malignancy. Immunol. Today 4, 211–213. Sasaki, J.-I., Dejehansart, M., and de Bruyn, J. (1994). The expression of mycobacterial heat-shock protein (HSP64) on Meth A tumour cells. Immunol. Cell Biol. 72, 415– 418. Sato, M., and Bremmer, I. (1993). Oxygen-free radicals and metallothionein. Free Radic. Biol. Med. 14, 325–337. Sato, M., and Sasaki, M. (1992). Tissue-specific induction of metallothionein synthesis by tumor necrosis factor a. Res. Commun. Chem. Pathol. Pharmacol. 75, 159 –172. Schroeder, J. J., and Cousins, R. J. (1990). Interleukin-6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures. Proc. Natl. Acad. Sci. U S A 87, 3137–3141. Sciavolino, P. J., Lee, T. H., and Vilcek, J. (1992). Overexpression of metallothionein confers resistance to the cytotoxic effect of TNF with cadmium in MCF-7 breast carcinoma cells. Lymphokine Cytokine Res. 11, 265–270. Srivastava, P. K., and Maki, R. G. (1991). Stress-induced proteins in immune response to cancer. Curr. Top. Microbiol. Immunol. 167, 109 –123.
208
YOUN AND LYNES
Srivastava, P. K., and Old, L. J. (1988). Individually distinct transplantation antigens of chemically induced mouse tumors. Immunol. Today 9, 78 – 83. Sugawara, S., Nowicki, M., Xie, S., Song, H.-J., and Dennert, G. (1990). Effects of stress on lysability of tumor targets by cytotoxic T cells and tumor necrosis factor. J. Immunol. 145, 1991–1998. Sugawara, S., Takeda, K., Lee, A., and Dennert, G. (1993. Suppression of stress protein GRP78 induction in tumor B/C10ME eliminates resistance to cell mediated cytotoxicity. Cancer Res. 53, 6001– 6005. Tagaya, Y., Okada, M., Sugie, K., Kasahara, T., Kondo, N., Hamuro, J., Matsushima, K., Dinarello, C. A., and Yodoi, J. (1988). IL-2 receptor (p55)/ Tac-inducing factor. Purification and characterization of adult T cell leukemia-derived factor. J. Immunol. 140, 2614 –2620. Thomas, D. G., Dingman, A. D., and Garvey, J. S. (1987). The function of metallothionein in cell metabolism. EXS 52, 539 –543. Thornally, P. J., and Vasak, M. (1985). Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta 827, 36 – 44. Tsangaris, G. T., and Tzortzatou-Stathopoulou, F. (1998). Metallothionein expression prevents apoptosis: A study with antisense phosphorothioate oligodeoxynucleotides in a human T-cell line. Anticancer Res. 18, 2423– 2433. Udono, H., and Srivastava, P. K. (1993). Heat shock protein 70-associated peptides elicit specific cancer immunity. J. Exp. Med. 178, 1391–1396. van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. (1996). Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science 274, 787–789. van Eden, W., Thole, J., van der Zee, R., Noordzij, A., van Embden, J. D. A.,
Hensen, E. J., and Cohen, I. R. (1988), Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis. Nature 331, 171–173. Wallich, R., Bulbuc, N., Hammerling, G. J., Katzav, S., Segal, S., and Feldman, M. (1985). Abrogation of metastatic properties of tumor cells by de novo expression of H-2K antigens following H-2 gene transfection. Nature 315, 301–305. Warner G. L., and Lawrence, D. A. (1988). The effect of metals on IL-2related lymphocyte proliferation. Int. J. Immunopharmacol. 10, 629 – 637. Whelan, M. A., and Archer, J. R. (1993). Chemical reactivity of an HLA-B27 thiol group. Eur. J. Immunol. 23, 3278 –3285. Wu, M., Lee, H., Bellas, R. E., Schauer, S. L., Arsura, M., Katz, D., FitzGerald, M. J., Rothstein, T. L., Sherr, D. H., and Sonenshein, G. E. (1996). Inhibition of NF-kappaB/Rel induces apoptosis of murine B cells. Embo J. 15, 4682– 4690. Yamauchi, A., Masutani, H., Tagaya, Y., Wakasugi, N., Mitsui, A., Nakamura, H., Inamoto, T., Ozawa, K., and Yodoi, J. (1992). Lymphocyte transformation and thiol compounds: The role of ADF/thioredoxin as an endogenous reducing agent. Mol. Immunol. 29, 263–270. Yoshino, I., Goedegebuure, P. S., Peoples, G. E., Lee, K.-Y., and Eberlein, T. J. (1994). Human tumor-infiltrating CD41 T cells react to B-cell lines expressing heat shock protein 70. J. Immunol. 153, 4149 – 4158. Youn, J., Borghesi, L. A., Olson, E. A., and Lynes, M. A. (1995). Immunomodulatory activities of extracellular metallothionein: II. Effects on macrophage functions. J. Toxicol. Environ. Health 45, 397– 413. Yurkow, E. J., and Makhijani, P. R. (1998). Flow cytometric determination of metallothionein levels in human peripheral blood lymphocytes: Utility in environmental exposure assessment. J. Toxicol. Environ. Health 54, 445– 457.
