Department of Molecular and Cell Biology, 75 North Eagleville Road, Unit 3125, University of Connecticut, Storrs, Connecticut 062693125
Received October 26, 2000; accepted February 28, 2001
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ABSTRACT |
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Key Words: metallothionein; humoral immunity; monoclonal anti-MT antibody; ovalbumin; isotype-matched antibody control..
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INTRODUCTION |
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Metallothionein (MT) is a small molecular weight (approximately 7 kDa), cysteine-rich stress response protein that binds heavy metals with high affinity (Hamer, 1986). Since the original characterization of MT as a cadmium-binding protein, there has been much interest in the structure and function of this protein (Vallee, 1995
). Although heavy metal cations such as cadmium, zinc, and copper are the most potent inducers of MT in mammals (Hamer, 1986
), a number of other agents can initiate increases in MT synthesis. These agents include free radicals (Iszard et al., 1995
, Klaassen and Lehman-McKeeman, 1989
), irradiation (Cai et al., 1999
; Morcillo et al., 2000
; Vukovic et al., 2000
), acute phase cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-
(TNF-
) (Cousins and Leinart, 1988
; Grider et al., 1989
; Sato et al., 1994
), inflammatory agents such as lipopolysaccharide (LPS) (Leibbrandt and Koropatnick, 1994
), and alkylating agents (Kotsonis and Klaassen, 1979
). MT is predominantly synthesized in liver (Coyle et al., 1995
; Quaife et al., 1999
), but other cells and tissues including lymphocytes, monocytes, and lymphoid tissues like the thymus can also produce MT under the appropriate stimuli (Coto et al., 1992
; Leibbrandt and Koropatnick, 1994
; Olafson, 1985
; Yurkow and Makhijani, 1998
).
Mammals express 4 major isoforms of MT. MT-I and MT-II are the primary inducible isoforms of MT, and can be expressed in most vertebrate tissues (Hamer, 1986). The expression of MT-III and MT-IV is more tissue specific. MT-III expression is associated with brain tissue (Palmiter et al., 1992
), and MT-IV is expressed predominantly in stratified squamous epithelium (Quaife et al., 1994
). Mice deficient in the expression of MT-I and MT-II have been produced by targeted disruption of the Mt1 and Mt2 genes. These mice have been used to determine the role of MT in the response to heavy metals (Masters et al., 1994
), UV irradiation (Michalska and Choo, 1993
; Reeve et al., 2000
), and to explore the roles of MT in immune function (Apostolova et al., 1997
; Crowthers et al., 2000
). MT-null mice have been shown to be highly sensitive to Cd-induced liver injury (Habeebu et al., 2000a
, b
; Liu et al., 2000
) and to Cd-induced hematoxicity and immunotoxicity (Liu et al., 1999
).
MT binds a number of metals including cadmium and mercury, so MT has been thought to play a protective role in cells and organisms exposed to heavy metals (Goering and Klaassen, 1984). MT can cause a shift in the subcellular distribution of toxic metals by removing them from nuclear fractions and can thereby reduce genotoxic damage (Goering and Klaassen, 1983
). Moreover, MT functions as a potent antioxidant by scavenging free radicals (Sato and Bremner 1993
; Thornalley and Vasak, 1985
). MT can also serve as a reservoir of essential metals, such as zinc and copper, which are required for growth and development (Daston et al., 1991
; De Lisle et al., 1996
; Zeng et al., 1991a
, b
).
Beyond these fundamental physiological roles, or perhaps as a consequence of these roles, MT has been shown to have several immunomodulatory effects. MT induces lymphocyte proliferation when added alone to splenocyte cultures, and can also act synergistically with other activators of B and T cells (e.g., LPS and Concanavalin A [Con A] [Lynes et al., 1990]) to stimulate cell division. MT can also enhance the division of cells that participate in an allogeneic mixed lymphocyte response (MLR) (Youn and Lynes, 1999
). While MT augments cell division, it can simultaneously reduce effector cell function. For example, cytotoxic T-cell killing of allogeneic targets is diminished in the presence of MT (Youn and Lynes, 1999
). Macrophages cultured in the presence of exogenous MT produce more superoxide anions, but support a weaker antigen specific T-cell response (Youn et al., 1995
).
MT can also decrease the in vivo humoral immune responses to T-dependent antigens. Injection of exogenous MT suppresses the specific anti-ovalbumin (OVA) response. Co-injected UC1MT (a monoclonal anti-MT antibody) blocks MT-mediated suppression of this anti-OVA response (Lynes et al., 1993).
The experiments described here are designed to explore the role of endogenous MT in humoral immunity. In these experiments we have examined the effect of UC1MT injection on the development of a humoral response to OVA challenge in the absence of exogenous MT. The results of these experiments demonstrate that endogenous MT can alter immune function. These observations may expand our understanding of how normal immune functions are regulated in the context of the inflammatory environment as well as to the immunomodulatory effects of a broad range of toxicants. Moreover, manipulation of MT levels with an anti-MT antibody may have important therapeutic implications in the context of toxicant-mediated immunosuppression, and in other instances where immune function is inadequate.
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MATERIALS AND METHODS |
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Media and reagents.
UC1MT (BALB/cJ IgG1 monoclonal anti-MT [Lynes et al., 1993], available from StressGen, Inc., Victoria, BC) was used in these experiments. UC1MT cells were maintained in culture in complete RPMI 1640 (GIBCO/BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT), 10 µM non-essential amino acids (GIBCO), 1 mM Na pyruvate (GIBCO), 1x BME vitamins (GIBCO), 50 µM 2-mercaptoethanol, penicillin/streptomycin (Sigma Chemical Co., St Louis, MO), and sodium bicarbonate. Cells were grown in the peritoneal cavity of CBySmn.CB17-Prkdcscid/J mice for the production of ascites. UC1MT antibody was purified by Protein-G affinity column chromotography according to manufacturer's instructions, and dialyzed against 0.85% NaCl. The isotype-matched control antibody (Mouse IgG1, kappa; MOPC 21) was purchased from Sigma and similarly prepared. Intact UC1MT and isotype control antibodies were used for in vivo treatments instead of F(ab')2 fragments of these antibodies to facilitate a prolonged presence of the antibody via FcRn-mediated recycling of the IgG (Junghans and Anderson, 1996
). UC1MT and MOPC 21 were labeled with FITC (5-fluorescein isothiocyanate, Eastman Kodak Company, Rochester, NY) according to previously described protocols (Holmes et al., 1995
).
Assessment of humoral responsiveness.
Mice were immunized via ip injection with 100 µg chicken egg OVA (Sigma). One-hundred µg purified UC1MT or 100 µg MOPC 21 was injected ip in treated groups of mice according to schedules described in the Results. All reagents were prepared in sterile 0.85% NaCl. Small samples of peripheral blood were drawn from the peri-orbital sinus starting at day 14 after the initial immunization, and serum was isolated and stored 20°C until analyzed. Experimental groups consisted of 5 age- and sex- matched mice.
ELISA.
Immulon 2 ELISA plates (Dynatech Laboratories, Inc., Alexandria, VA) were used for these assays. Antigen (OVA) was diluted in coating buffer (1.57% Na2CO3, 2.93% NaHCO3, 0.2% sodium azide, pH 9.7). The plates were incubated with 100 µl antigen/well for 1 h at 37°C. The plates were aspirated and subsequent nonspecific protein absorption was blocked with 200 µl of 1% Teleostean gelatin (Sigma) in phosphate buffered saline (PBS) with Tween 20 and NaN3. After a 1-h incubation at 37°C, the plates were washed 3 times with wash buffer (PBS with 0.2% NaN3 and 0.005% Tween 20, pH 7.2) in a BioTek EL403 automated plate washer. One-hundred µl of mouse serum diluted in 1% BSA (1/500 dilution) was then added to individual wells. Following a 2-h incubation at 37°C, the plates were washed with wash buffer and incubated for 1 h at 37°C with secondary antibody (alkaline phosphatase conjugates) in 1% BSA. Finally, the wells were washed and 100 µl of substrate (1 mg/ml of para-nitrophenyl phosphate in DEA buffer: 9.7% diethanolamine, 0.02% NaN3, and 0.01% MgCl2, pH 9.8) was added to each well. Kinetic color development was determined in a Tmax ELISA microplate reader (Molecular Devices, Menlo Park, CA) at 405 nm.
Total immunoglobulin levels were also determined by using ELISA. Briefly, Immulon 2 ELISA plates were coated with 100 µl of capture antibody (goat anti-mouse Ig [H + L], Southern Biotechnology, Birmingham, AL) in coating buffer and incubated overnight at 4°C. The plates were then blocked with 2% BSA in coating buffer for 1 h at 37°C. After incubation, the plates were washed and 100 µl of purified immunoglobulin at known concentrations or serum samples from experimental animals diluted in 1% BSA in PBS were added to appropriate wells. The plates were incubated for 1 h at 37°C, then washed and incubated with goat anti-mouse IgG or IgM specific antibodies conjugated to alkaline phosphatase (Southern Biotechnology). Color development and measurement was performed as described in the previous section.
ELISPOT assay.
The ELISPOT (Enzyme-Linked Immunospot) assay can be used to determine the number of antibody-secreting cells in a given number of splenocytes (Czerkinsky et al., 1983; Moller and Borrebaeck, 1985
). Briefly, sterile 96-well filtration plates with surfactant-free mixed cellulose ester membrane (Millipore Corp., Bedford, MA) were coated with OVA (100 µg/ml) in coating buffer. Control wells were incubated with 100 µg/ml nonfat dry milk. The plates were incubated overnight at 4°C and washed the following day with PBS/azide using an automated plate washer. Nonspecific binding was blocked with 1% Teleostean gelatin in PBS. Single cell suspensions from spleens of immunized mice were prepared in complete RPMI 1640 (starting at 1 x 106 cells/well) and were added to appropriate wells. After overnight incubation at 37°C in humidified 5% CO2 incubator, cells were removed from the plate and wells were washed 3 times with PBS with azide. The plates were then incubated with secondary antibody (goat anti-mouse IgG conjugated to alkaline phosphatase) for 2 h at room temperature and washed again with PBS with azide. Spot-forming cells were detected by the addition of 100 µl of BCIP/NBT substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) to each well at room temperature. After the formation of visible spots, the reaction was stopped by washing the plate several times with ddH2O, and spots were counted under the microscope after coding the wells.
Flow cytometry.
To determine the expression of cell surface antigens, splenocytes (1 x 106 cells/well) depleted of erythrocytes by hypotonic lysis were obtained from immunized mice and were pre-incubated with 10% goat serum (GIBCO) in PBS for 1 h. After this incubation, cells were washed twice with FACS buffer (PBS, 5% FBS, 0.1% NaN3) and incubated with conjugated anti-mouse IgG-FITC (ImmunoPure, PIERCE, Rockford, IL), anti-mouse IgG + IgM-FITC (Tago Inc., Burlingame, CA), anti-mouse CD4-FITC (PharMingen, Becton Dickinson Co., San Diego, CA), or anti-mouse CD8-APC (PharMingen) for 30 min. After another series of washes, analysis was performed with a Becton Dickinson FACSCalibur (Mountain View, CA) using Cellquest 3.2.1 application software.
MT binding to splenocytes.
Splenocytes from BALB/cByJ mice were incubated in mixed gas (10% CO2, 7% O2, 83% N2) in the absence or presence of MT (20 µM) for 15 or 40 h. After incubation, splenocytes were counted and diluted in FACS buffer (PBS, 5% FBS, and 0.1% NaN3). For flow cytometry analysis, nonspecific binding to splenocytes (1 x 106 cells/well) was blocked by incubation with rabbit IgG (Sigma) in PBS on ice for 45 min. Then, cells were washed with FACS buffer and incubated with appropriate dilutions of UC1MT-FITC or MOPC 21-FITC on ice for 40 min. After washing the cells 3 times with FACS buffer, analysis was performed with a FACSCalibur. In addition, splenocytes from C57BL/6J-Hcphmev mev/mev and +/mev mice were used in some experiments. C57BL/6J-Hcphmev mev/mev mice have been reported to have elevated levels of serum MT (Lynes et al., 1999). Splenocytes from both types of mice were incubated with UC1MT-FITC in the absence of exogenous MT to determine the presence of MT on the surface of naive autoimmune splenocytes.
Statistical analysis.
The Student's t-test was used to determine significant differences between control and treated group of mice. Differences were considered statistically significant when p < 0.05.
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RESULTS |
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DISCUSSION |
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Injection of UC1MT monoclonal antibody enhances the antigen-specific response to OVA challenge, as shown by increased levels of anti-OVA antibody and by increases in the number of OVA-specific plasma cells. The lack of effect of UC1MT on total serum antibody levels and on the general T- and B-cell populations suggests that the cells most affected are those that receive an antigenic challenge concurrent with UC1MT administration.
The pattern of immune enhancement after UC1MT treatment is interesting. The humoral response to OVA is predominantly of the IgG1 isotype. In mice, the IgG1 and IgG2a responses are regulated by distinct helper T-cell subsets. IgG1 usually depends upon Th2 cells, which secrete cytokines including IL-4, IL-5, and IL-10, and serve to stimulate the growth and differentiation of B cells into plasma cells. In contrast, IgG2a production is dependent on the activity of Th1 cells, which secrete cytokines including IL-2 and -IFN (Romagnani, 1997
). Significant increases in the levels of OVA-specific IgG1 following UC1MT treatment suggests that MT may act to suppress the function of Th2 cells.
These results are in concordance with several previously reported observations. Exogenous MT, co-injected with challenge antigen, was found to suppress the T-dependent humoral immune response (Lynes et al., 1993). In those experiments, UC1MT was able to block the action of the exogenous MT. Exogenous MT has also been found to suppress T-dependent cytotoxic responses (Youn and Lynes, 1999
). Results reported here are also compatible with the observation that targeted gene disruptions of the endogenous metallothionein-1 (Mt1) and metallothionein-2 (Mt2) genes in mice result in an animal with an elevated humoral response to T-dependent antigen challenge (Crowthers et al., 2000
). Other groups have also remarked on the interplay between MT and the immune system. For example, MT has been found to be expressed in the thymic tissues (Mocchegiani et al., 1998
; Olafson, 1985
; Savino et al., 1984
) and MT expression in this tissue has been suggested to play an essential role in the functional activities of the immature thymocyte. Peripheral blood lymphocytes have also been shown to express MT (Vandeghinste et al., 2000
). One of the potential roles that MT may play in these contexts is as a pro-apoptotic element (Houben et al., 1997
), which could contribute to the immunosuppressive effects of MT that we have observed.
Our results are interesting from a number of different perspectives. First, they relate to the regulatory mechanisms that operate during a normal immune response. We hypothesize that MT can be synthesized within the microenvironment of an activated immune response as a consequence of agents produced by the activated response. These inducers include several of the acute phase cytokines, but IL-6 appears to have a central role in the activation of MT synthesis (Choudhuri et al., 1994; Itoh et al., 1994
; Penkowa and Hidalgo, 2000
; Schroeder and Cousins, 1990
). MT synthesis may also result from the local inflammatory oxidative environment that occurs in the context of macrophage, monocyte, and neutrophil activation (Backman et al., 1998
; Leibbrandt and Koropatnick, 1994
), or as a consequence of the cellular stress that occurs as cells respond to proliferative signals (Arora et al., 1998
; De et al., 1990
).
Once synthesized, MT may remain within the cell, and can compartmentalize to either the nucleus or the cytosol (Cherian 1994; Kondo et al., 1995
) but it can also be found outside the cell. As an intracellular protein, MT may serve to sequester essential heavy metals such as zinc and copper that are needed for a variety of enzymatic activities and transcription regulators (Zeng et al., 1991b
) related to proliferation and differentiation (Daston et al., 1991
; Koterov et al., 1996
). MT may also act as an anti-oxidant to protect cellular machinery from damage that would otherwise occur as a consequence of oxidant exposure, but this anti-oxidant effect may also serve to decrease oxidants that themselves function as elements of the signal transduction cascade. MT may also negatively regulate gene activity by direct interactions with transcription factors like NF-
B (Abdel-Mageed and Agrawal, 1998
; Sakurai et al., 1999
). As an extracellular protein, MT has been found in a variety of locations including the pancreatic ducts (De Lisle et al., 1996
), bronchoalveolar spaces (Hart and Garvey, 1986
), urine (Kido et al., 1991
), and serum (Hidalgo et al., 1986
; Thomas et al., 1986
). There is evidence that other stress-response proteins can also be selectively released from cells (Hightower and Guidon, 1989
). This extracellular pool of MT can interact with the surfaces of cells. Wild-type splenocytes placed in culture for 15 or 40 h are bound by UC1MT monoclonal anti-MT antibody, albeit at low levels (Figs. 5A and 5B
), while control cells from naive wild-type mice are not labeled with anti-MT antibody immediately following harvest from the spleen (Fig. 6
). This UC1MT-labeling of cultured cells may reflect the induction of MT by oxidative stress that is associated with standard culture conditions (Lawrence et al., 1996
). Incubation of wild-type cells with exogenous MT increases the binding of UC1MT to these cell surfaces, underscoring the observation that MT can bind to the cell surface, as does the use of a biotinylated form of MT to show binding to the lymphocyte plasma membrane (Borghesi et al., 1996
). MT has been found in the circulation of viable motheaten (Hcphmev) mice (Lynes et al., 1999
), and we have found that naïve splenocytes from this autoimmune animal are labeled with the UC1MT antibody at levels significantly above wild-type controls (Fig. 6
). The binding of MT to the plasma membrane may be non-specific, but there is one report of a MT-specific receptor on astrocytes (El Refaey et al., 1997
). Similar receptors might be present on hematopoietic cells.
The binding of MT to cell surfaces can alter the ability of those cells to function under certain circumstances. While MT increases the proliferative response to polyclonal activators (Borghesi et al., 1996), it also reduces the differentiation of effector lymphocytes (Youn and Lynes, 1999
). Anti-MT antibody may serve to disrupt this suppressive effect of extracellular MT on immune functioning, releasing the immune system to produce a more vigorous response. In this context, individuals that develop an inflammatory autoimmune disease in the absence of adequate MT levels may experience a more severe form of the disease than they might in the presence of MT.
Viable motheaten animals that have been engineered to have disrupted Mt1 and Mt2 genes experience a more severe disease than animals with normal MT genes (Lynes et al., 1999). Additionally, in a murine model of arthritis, injection of collagen normally produces dramatic joint swelling, but simultaneous administration of exogenous MT blocks this disease process (Youn, personal communication). There are several reports that serum MT levels are altered in human patients with certain forms of autoimmune disease. For example, serum MT levels are decreased below normal in individuals with rheumatoid arthritis and systemic lupus erythematosus (Miesel and Zuber, 1993a
, b
). The authors suggest that MT is depleted in these patients as a consequence of inflammation-associated oxidation, but it may also be that patients diagnosed with the most severe cases of autoimmune disease are those who are least able to synthesize adequate amounts of this essential protein. Other reports note elevated MT in patients with various forms of autoimmunity, including arthritis and amyotrophic lateral sclerosis (Backman et al., 1998
; Coyle et al., 1995
; Sillevis Smitt et al., 1994
; Winters et al., 1997
). Little work has been done to characterize the range of MT synthesis potential in the human population, but there are suggestions that the range can be substantial (Yurkow and Makhijani, 1998
).
We hypothesize that MT may be released from cells exposed to a stressful environment as a negative regulator of the immune response, preventing the immune response from exceeding some useful limit while simultaneously moderating the oxidative environment. It is possible that in each of these instances, one role that MT plays is to suppress the autoimmune attack on self tissues, serving to limit the severity of the disease. It is intriguing to speculate that a therapeutic increase in MT synthesis levels may be advantageous to patients with autoimmune disease. The induction of MT by glucocorticoids and by colloidal gold salts may represent an aspect of the therapeutic mechanisms initiated by these treatments of autoimmune disease. On the other hand, MT may have deleterious effects under other circumstances. A number of reports have shown that various neoplastic cell lines and tissues synthesize MT in elevated amounts (Cherian et al., 1993; Ebadi and Iversen, 1994
). Under these circumstances, MT released to the circulation may suppress desirable anti-tumor reactivity as well as enhancing the viability of the tumor within the oxidative environment of the tumor.
Exposure to a variety of environmental toxicants have also been reported to elicit MT synthesis and to be immunosuppressive. As already noted, toxicant exposure can result in elevated serum MT (Nakashima et al., 1997). In light of the results reported here, it is reasonable to hypothesize that the MT synthesized as a consequence of that toxicant exposure may be in part responsible for the immunosuppression associated with toxicant exposure. Moreover, it is possible that judicious treatment with a species-appropriate monoclonal anti-MT antibody may restore normal immune function in the case of toxicant-exposure, and might actually enhance desirable immune function.
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ACKNOWLEDGMENTS |
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NOTES |
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