@Copyright 1986 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0163-4984/86/110(M)129503.90
In Vitro Regulation of Human Lymphocyte Proliferation by Selenium HOWARD T. PETRIE,* LYNELL W. KLASSEN, MARGARET A. TEMPERO," AND H. DAVID KAY Experimental Immunology Laboratory, Section of Rheumatology and Immunology, a n d aSection of Ontology and Hematology, Department of Internal Medicine, Omaha VeteransAdministration Medical Center and University of Nebraska Medical Center, Omaha, Nebraska 68105, USA Received J u n e 30, 1986; Accepted S e p t e m b e r 9, 1986.
ABSTRACT A chemoprotective role for dietary selenium in malignancy has been well documented in numerous epidemiological and experimental studies. The precise mechanisms of this relationship are not understood, but may be related to observations that selenium can inhibit the proliferation of various normal and neoplastic cells, both in vivo and in vitro. In this study, we present evidence that selenium at physiologic concentrations can effectively inhibit the overall proliferation of human lymphocyte populations in response to various immune stimuli in vitro, including mixed lymphocyte response and response to soluble antigen (tetanus toxoid). This inhibition was reversible, indicating that selenium was not toxic to the lymphocytes at these concentrations. Preliminary data from our laboratory indicate that the antiproliferative effects of selenium may be specific for certain lymphocyte subsets. Similar modulation of immune responses in vivo could enhance various humoral and cellular immune mechanisms. Together with published evidence that selenium can inhibit tumor cell proliferation, these data may help to explain the decreased incidence of cancer associated with elevated selenium intake. *Author to whom all correspondence and reprint requests should be addressed, at: Research Service, R321, Omaha Veterans Administration Medical Center, 4101 Woolworth Ave., Omaha NE 68105 USA. Biological
Trace Element Research
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Index Entries: Lyrnphocytes, regulation of proliferation by selenium; selenium, regulation of proliferation by; proliferation, inhibition by selenium; immunity, regulation of.
INTRODUCTION An inverse correlation between cancer mortality and dietary intake of the trace mineral element selenium has been well documented in a number of epidemiologic studies in humans and experimental studies in animals (1-7). The precise mechanism(s) of this association are still undetermined, although a number of explanations have been proposed. For one, selenium is known to be an essential cofactor for the enzyme glutathione peroxidase (GSH-Px), which functions intracellularly to detoxify highly reactive oxygen radicals that can cause damage to DNA. Elevating selenium levels in body tissues could therefore elevate GSH-Px activity in tissues and thereby enhance its DNA protective effects. However, several detailed experimental studies (4,5,7,8) have demonstrated that the observed chemoprotective effects of selenium are not completely attributable to the elevation or maintenance of high levels of GSH-Px activity. It has also been proposed that the chemoprotective effect of selenium may be the result of the prevention by selenium of the production of carcinogenic metabolites from their noncarcinogenic precursors (9), although not all investigators agree with this concept (10). In any case, this cannot be the sole mechanism since it has been shown that selenium can also prevent the induction of cancer by carcinogens that do not require metabolic activation (5). Thompson et al. (7) have demonstrated that selenium can block both the initiation of neoplasia and the subsequent events involved in promotion of the tumor. Whether selenium functions differentially in initiation, promotion, or expression of tumors is still unknown; selenium has since been demonstrated in various models to be effective both early (11,12) and late (8,12) in the various stages of carcinogenesis. Most recently, attention has focused on observations that elevated levels of selenium have been shown to cause the inhibition of cellular proliferation of various cell types, both in vivo and in vitro. Dietary selenium supplementation in mice and rats has decreased the growth rate of both normal cells (8,13-15) and neoplastic cells, induced in vivo or transplanted (8,13,16-20). In addition, selenium supplementation of culture media has been shown to inhibit the in vitro growth of various transformed cell types (8,13,14,19). In contrast to these inhibitory effects of selenium, elevated selenium intake causes the enhancement of diverse immune responses in experimental animals (21-25). Dietary selenium supplementation has been shown to enhance production of IgG and IgM anti-sheep red blood cell (SRBC) antibodies in immunized mice (21), and selenium administered at the same time as SRBC has been shown to function as an immunoadjuBiological Trace Element Research
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vant (26). Natural killer (NK) cell cytotoxic activity has been shown to be enhanced in rats supplemented with selenium in their diets as compared with rats on normal diets (22,25). The modulation of other immune responses and/or immunoregulatory molecules has also been described
(25). The concurrent expression of enhanced immunity and decreased tumor cell proliferation in response to elevated selenium levels could be expected to play a significant role in the reduction of tumor incidence (27). However, very little is known regarding the effects of selenium on human immunity. To date, only two studies have been published in this regard (24,28). The studies reported here are designed to help to fill this gap, and to provide some of the first data oil the effects of selenium on human immune responses in vitro. Lymphoproliferation and the subsequent differentiation of human peripheral blood lymphocytes (PBL) in vitro after antigenic challenge is a well documented phenomenon. This event can be initiated by soluble or cellular foreign antigens and leads to the generation of clonally expanded, mature, functional immune cells specific for and directed against the original stimulus. Since both proliferation and subsequent differentiation and maturation are required for this reactivity, substances that can regulate any aspect of this process might be expected to influence the ultimate rate and/or magnitude of these immune functions. In this study, we report that selenium can effectively regulate the proliferation of iymphocytes in several in vitro culture systems and propose potential mechanisms for the role of immunity in selenium-reduced cancer incidence.
MATERIALS AND METHODS Lymphocytes Mononuclear peripheral blood lymphocytes (PBL) were isolated from the peripheral blood of normal volunteer donors using Lymphocyte Separation Medium (LSM, Litton Bionetics Inc., Charleston SC, USA), as described (29). Mononuclear cell preparations were maintained in RPMI 1640 solution supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES buffer, 2 mM glutamine, 1 mM sodium pyruvate, 1• minimal essential medium vitamins, and lX minimal essential medium amino acids (all reagents from KC Biological, Lenexa, KS, USA), with 50 tzg/mL of gentamycin sulfate (Sigma Chemical Co., St. Louis, MO, USA). This enriched culture medium was used in all experiments and will subsequently be referred to simply as medium.
Selenium A stock solution of sodium selenite (Na2SeO3) (5 x 10-4M), dissolved in RPMI 1640 solution, was used as the starting solution for all Biological Trace Element Research
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experiments described. Further dilutions were made as appropriate in medium.
/Vlixed Lymphocyte Cultures One-way mixed lymphocyte cultures (IXMLC) were generated in 96-well flat-bottomed plates as follows: 100 ILL of responding PBL at 2 x 10 6 cells/mL in medium were mixed with an equal volume of gammairradiated (2Krad) allogeneic stimulating PBL at 4 x 106 cells/mL of medium in quadruplicate replicates. To appropriate replicate wells, 100 p~L of various dilutions of 3 • concentrated sodium selenite solution were added (300 ~L final volume). Control cultures received 100 ~L of medium alone. Proliferation of responding cells was measured on the days indicated, using 3H-thymidine, as described below.
Tetanus Antigen Stimulation Proliferative response of PBL to tetanus antigen was determined in 96-well plates as follows: 100 p,L of responding PBL at 2 x 10~ cells/mL of medium were mixed with 100 p~L of tetanus toxoid solution (Lederle Laboratories, Pearl River NY, USA) in medium in quadruplicate replicates. Desired levels of selenium were obtained by adding 100 ~L of various dilutions of 3x concentrated sodium selenite solution to appropriate wells (300 ~L final volume). Control cultures received 100 ~L of medium alone. Final dilution of tetanus antigen was 1:5000. Proliferation of the cells in the stimulated culture was measured on the days indicated, using 3H-thymidine, as described below.
31-1-Thymidine Assay Proliferative levels of lymphocytes stimulated in vitro were determined by the capacity of these cells to incorporate radiolabeled thymidine into newly synthesized DNA. Four hours before the desired harvest time, 1 IJ,Ci of methyl-3H-thymidine (Research Products International Corp., Mt. Prospect IL, USA) in 10 IJ,L of RPMI 1640 solution was added to each experimental well of flat-bottomed 96-well tissue culture plates (Corning Glass Co., Corning NY, USA). Plates were incubated at 37~ under humidified conditions of 5% CO2/air for 4 h. Radiolabeled cultures were harvested using a mini-MASH apparatus (MA Bioproducts, Walkersville MD, USA). Radioactivity was measured using a Beckman LS1800 liquid scintillation counter. All assays were performed in quadruplicate replicates.
Kinetics of the Inhibition of MLC by Selenium The MLC were generated in 96-well plates, as described above, with the following modifications: all wells received 100 ~L of medium in addi-
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tion to responding and stimulating PBL (300 ILL final volume). At various times, ranging from 0 to 6 d postallogeneic stimulation, 33.3 p,L of 10 x concentrated selenium (5 • 10-~M in medium) were added to culture wells (5 • 10-6M Se final concentration, 333.3 p,L final culture volume). On d 6, cultures were pulsed with 3H-thymidine and harvested as described above.
Recovery of MLC Proliferation Postselenium Treatment Two 25-cm2 tissue-culture flasks were each inoculated with 1 • 107 responding PBL and 2 x 10 7 gamma-irradiated stimulating PBL in 6 mL medium. One flask received 36 p,L of 5 • 10- 4M Se stock solution (3 • 10 6M Se final concentration); the other flask was untreated (control). Both flasks were incubated upright at 37~ for 8 h under humidified conditions of 5% CO2/air. Following the treatment period, 5 mL of the supernatant medium were carefully aspirated from each culture and replaced with 14 mL of flesh medium (15 mL final volume). After 24 h of culture, 12 mL of culture supernatant were again removed and replaced with fresh medium. On d 4, 6, and 8 of incubation, cultures were resuspended, and 200 ILL from each flask were dispensed into each of four wells of 96-well flat-bottomed plates. Proliferative levels in each culture were determined from these wells using 3H-thymidine as described above. In addition, 20 lzL were removed from each flask for analysis of cell numbers by Coulter counting.
Recovery of Phytohemagglutinin-lnduced Proliferation Postselenium Treatment Five 25-cm2 tissue-culture flasks were each inoculated with 2.5 mL of PBL at 2 x 106/mL in medium. Appropriate concentrations of 2 x concentrated selenium were then added in 2.5 mL of medium (5 mL final volume). After overnight incubation at 37~ cell suspensions were removed from flasks, washed 2• in medium to remove free Se, resuspended in 5 mL of medium, and incubated at 37~ for an additional 24 h. Following the second incubation, the cell suspensions were washed again, resuspended in 5 mL of medium, and 100 p,L of each culture were then dispensed into quadruplicate wells of 96-well plates. To each well, 100 FL of medium containing phytohemagglutinin (PHA, 2.0 ~g/mL, Miles Laboratories, Elkhart IN, USA) were added. Plates were incubated at 37~ for 3 d, and proliferation of cells was then measured using 3H-thymidine, as described above.
Cell Plumbers in Selenium-Treated ]vlLC One-way MLC were set up as described under mixed lymphocyte cultures (above). After 7 d of culture, 10 ~L of Hematall LA-Hgb lysing
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reagent (Fisher Scientific, Fairlawn NJ, USA) were added to each well. Twenty microliters from each well were then counted using a Coulter Counter (Coulter Electronics, Hialeah, FI, USA).
Normalization of 3H- Thymidine Incorporation Values The mean value in counts per minute (cpm) for 3H-thymidine incorporation by stimulated control cultures (no selenium) was calculated by pooling the control values for all individual donors (pooled control mean). A normalization factor was then calculated separately for each donor by dividing the pooled control mean by the control mean for that donor. Normalized values for each experimental set were calculated by multiplying the experimental mean of that set times the normalization factor for that donor.
RESOLTS Inhibition of Lymphocyte Proliferation in MLC The addition of selenium to lymphocytes stimulated to proliferate in one-way MLC resulted in a d o s e - d e p e n d e n t inhibition of proliferation (Fig. 1). The onset of inhibitory concentrations of selenium occurred in the range of 2-3 x 10- 4M, d e p e n d i n g on the time in culture. The statistical significance of inhibition by 2 • 10 ('M selenium was p < 0.001 on d 4 of culture, p < 0.1 on d 6, and not statistically significant on d 8, as analyzed by the Student's t-test for paired samples. Inhibition by selenium at 3 • 10 6M concentration and higher was statistically significant at the p < 0.001 level on all days of culture measured.
Inhibition of Lymphocyte Proliferation in Response to Tetanus Antigen When a d d e d to lymphocyte cultures, which had been stimulated with tetanus toxoid, 2 • 10 6M selenium showed highly significant levels of inhibition of proliferation (Fig. 2) on d 5 and 7 (p < 0.05) and less significant inhibition on d 9 (p < 0.1). Inhibition by 3 • 10 6M selenium was significant on all days tested (p < 0.02 on d 5 and 7 and p < 0.05 on d 9).
Recovery of Proliferative Capacity After Selenium Pretreatment The observed inhibition of lymphocyte proliferation by selenium could be interpreted as indicating that selenium at these concentrations was toxic to the responding lymphocytes; nonviable lymphocytes would be incapable of proliferation. Nonviability is defined in this case to mean a permanent and irreversible loss of proliferative capacity. In order to investigate this possibility, we incubated MLC for 8 h with a concentration Biolooical Trace Element Research
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[S4x lo-' M Fig. 1. Inhibition of MLC proliferation by selenium. When added to 1XMLC at the time of allogeneic stimulation, selenium caused the dose-dependent inhibition of proliferative response as measured by BH-thymidine incorporation. Inhibition was statistically significant in the range of 2-3 x 10
In Vitro Regulation of Human Lymphocyte Proliferation by Selenium HOWARD T. PETRIE,* LYNELL W. KLASSEN, MARGARET A. TEMPERO," AND H. DAVID KAY Experimental Immunology Laboratory, Section of Rheumatology and Immunology, a n d aSection of Ontology and Hematology, Department of Internal Medicine, Omaha VeteransAdministration Medical Center and University of Nebraska Medical Center, Omaha, Nebraska 68105, USA Received J u n e 30, 1986; Accepted S e p t e m b e r 9, 1986.
ABSTRACT A chemoprotective role for dietary selenium in malignancy has been well documented in numerous epidemiological and experimental studies. The precise mechanisms of this relationship are not understood, but may be related to observations that selenium can inhibit the proliferation of various normal and neoplastic cells, both in vivo and in vitro. In this study, we present evidence that selenium at physiologic concentrations can effectively inhibit the overall proliferation of human lymphocyte populations in response to various immune stimuli in vitro, including mixed lymphocyte response and response to soluble antigen (tetanus toxoid). This inhibition was reversible, indicating that selenium was not toxic to the lymphocytes at these concentrations. Preliminary data from our laboratory indicate that the antiproliferative effects of selenium may be specific for certain lymphocyte subsets. Similar modulation of immune responses in vivo could enhance various humoral and cellular immune mechanisms. Together with published evidence that selenium can inhibit tumor cell proliferation, these data may help to explain the decreased incidence of cancer associated with elevated selenium intake. *Author to whom all correspondence and reprint requests should be addressed, at: Research Service, R321, Omaha Veterans Administration Medical Center, 4101 Woolworth Ave., Omaha NE 68105 USA. Biological
Trace Element Research
] 29
rot 1 I, 1986
Petrie et al.
130
Index Entries: Lyrnphocytes, regulation of proliferation by selenium; selenium, regulation of proliferation by; proliferation, inhibition by selenium; immunity, regulation of.
INTRODUCTION An inverse correlation between cancer mortality and dietary intake of the trace mineral element selenium has been well documented in a number of epidemiologic studies in humans and experimental studies in animals (1-7). The precise mechanism(s) of this association are still undetermined, although a number of explanations have been proposed. For one, selenium is known to be an essential cofactor for the enzyme glutathione peroxidase (GSH-Px), which functions intracellularly to detoxify highly reactive oxygen radicals that can cause damage to DNA. Elevating selenium levels in body tissues could therefore elevate GSH-Px activity in tissues and thereby enhance its DNA protective effects. However, several detailed experimental studies (4,5,7,8) have demonstrated that the observed chemoprotective effects of selenium are not completely attributable to the elevation or maintenance of high levels of GSH-Px activity. It has also been proposed that the chemoprotective effect of selenium may be the result of the prevention by selenium of the production of carcinogenic metabolites from their noncarcinogenic precursors (9), although not all investigators agree with this concept (10). In any case, this cannot be the sole mechanism since it has been shown that selenium can also prevent the induction of cancer by carcinogens that do not require metabolic activation (5). Thompson et al. (7) have demonstrated that selenium can block both the initiation of neoplasia and the subsequent events involved in promotion of the tumor. Whether selenium functions differentially in initiation, promotion, or expression of tumors is still unknown; selenium has since been demonstrated in various models to be effective both early (11,12) and late (8,12) in the various stages of carcinogenesis. Most recently, attention has focused on observations that elevated levels of selenium have been shown to cause the inhibition of cellular proliferation of various cell types, both in vivo and in vitro. Dietary selenium supplementation in mice and rats has decreased the growth rate of both normal cells (8,13-15) and neoplastic cells, induced in vivo or transplanted (8,13,16-20). In addition, selenium supplementation of culture media has been shown to inhibit the in vitro growth of various transformed cell types (8,13,14,19). In contrast to these inhibitory effects of selenium, elevated selenium intake causes the enhancement of diverse immune responses in experimental animals (21-25). Dietary selenium supplementation has been shown to enhance production of IgG and IgM anti-sheep red blood cell (SRBC) antibodies in immunized mice (21), and selenium administered at the same time as SRBC has been shown to function as an immunoadjuBiological Trace Element Research
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Selenium Regulation of Lymphoproliferation
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vant (26). Natural killer (NK) cell cytotoxic activity has been shown to be enhanced in rats supplemented with selenium in their diets as compared with rats on normal diets (22,25). The modulation of other immune responses and/or immunoregulatory molecules has also been described
(25). The concurrent expression of enhanced immunity and decreased tumor cell proliferation in response to elevated selenium levels could be expected to play a significant role in the reduction of tumor incidence (27). However, very little is known regarding the effects of selenium on human immunity. To date, only two studies have been published in this regard (24,28). The studies reported here are designed to help to fill this gap, and to provide some of the first data oil the effects of selenium on human immune responses in vitro. Lymphoproliferation and the subsequent differentiation of human peripheral blood lymphocytes (PBL) in vitro after antigenic challenge is a well documented phenomenon. This event can be initiated by soluble or cellular foreign antigens and leads to the generation of clonally expanded, mature, functional immune cells specific for and directed against the original stimulus. Since both proliferation and subsequent differentiation and maturation are required for this reactivity, substances that can regulate any aspect of this process might be expected to influence the ultimate rate and/or magnitude of these immune functions. In this study, we report that selenium can effectively regulate the proliferation of iymphocytes in several in vitro culture systems and propose potential mechanisms for the role of immunity in selenium-reduced cancer incidence.
MATERIALS AND METHODS Lymphocytes Mononuclear peripheral blood lymphocytes (PBL) were isolated from the peripheral blood of normal volunteer donors using Lymphocyte Separation Medium (LSM, Litton Bionetics Inc., Charleston SC, USA), as described (29). Mononuclear cell preparations were maintained in RPMI 1640 solution supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES buffer, 2 mM glutamine, 1 mM sodium pyruvate, 1• minimal essential medium vitamins, and lX minimal essential medium amino acids (all reagents from KC Biological, Lenexa, KS, USA), with 50 tzg/mL of gentamycin sulfate (Sigma Chemical Co., St. Louis, MO, USA). This enriched culture medium was used in all experiments and will subsequently be referred to simply as medium.
Selenium A stock solution of sodium selenite (Na2SeO3) (5 x 10-4M), dissolved in RPMI 1640 solution, was used as the starting solution for all Biological Trace Element Research
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experiments described. Further dilutions were made as appropriate in medium.
/Vlixed Lymphocyte Cultures One-way mixed lymphocyte cultures (IXMLC) were generated in 96-well flat-bottomed plates as follows: 100 ILL of responding PBL at 2 x 10 6 cells/mL in medium were mixed with an equal volume of gammairradiated (2Krad) allogeneic stimulating PBL at 4 x 106 cells/mL of medium in quadruplicate replicates. To appropriate replicate wells, 100 p~L of various dilutions of 3 • concentrated sodium selenite solution were added (300 ~L final volume). Control cultures received 100 ~L of medium alone. Proliferation of responding cells was measured on the days indicated, using 3H-thymidine, as described below.
Tetanus Antigen Stimulation Proliferative response of PBL to tetanus antigen was determined in 96-well plates as follows: 100 p,L of responding PBL at 2 x 10~ cells/mL of medium were mixed with 100 p~L of tetanus toxoid solution (Lederle Laboratories, Pearl River NY, USA) in medium in quadruplicate replicates. Desired levels of selenium were obtained by adding 100 ~L of various dilutions of 3x concentrated sodium selenite solution to appropriate wells (300 ~L final volume). Control cultures received 100 ~L of medium alone. Final dilution of tetanus antigen was 1:5000. Proliferation of the cells in the stimulated culture was measured on the days indicated, using 3H-thymidine, as described below.
31-1-Thymidine Assay Proliferative levels of lymphocytes stimulated in vitro were determined by the capacity of these cells to incorporate radiolabeled thymidine into newly synthesized DNA. Four hours before the desired harvest time, 1 IJ,Ci of methyl-3H-thymidine (Research Products International Corp., Mt. Prospect IL, USA) in 10 IJ,L of RPMI 1640 solution was added to each experimental well of flat-bottomed 96-well tissue culture plates (Corning Glass Co., Corning NY, USA). Plates were incubated at 37~ under humidified conditions of 5% CO2/air for 4 h. Radiolabeled cultures were harvested using a mini-MASH apparatus (MA Bioproducts, Walkersville MD, USA). Radioactivity was measured using a Beckman LS1800 liquid scintillation counter. All assays were performed in quadruplicate replicates.
Kinetics of the Inhibition of MLC by Selenium The MLC were generated in 96-well plates, as described above, with the following modifications: all wells received 100 ~L of medium in addi-
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tion to responding and stimulating PBL (300 ILL final volume). At various times, ranging from 0 to 6 d postallogeneic stimulation, 33.3 p,L of 10 x concentrated selenium (5 • 10-~M in medium) were added to culture wells (5 • 10-6M Se final concentration, 333.3 p,L final culture volume). On d 6, cultures were pulsed with 3H-thymidine and harvested as described above.
Recovery of MLC Proliferation Postselenium Treatment Two 25-cm2 tissue-culture flasks were each inoculated with 1 • 107 responding PBL and 2 x 10 7 gamma-irradiated stimulating PBL in 6 mL medium. One flask received 36 p,L of 5 • 10- 4M Se stock solution (3 • 10 6M Se final concentration); the other flask was untreated (control). Both flasks were incubated upright at 37~ for 8 h under humidified conditions of 5% CO2/air. Following the treatment period, 5 mL of the supernatant medium were carefully aspirated from each culture and replaced with 14 mL of flesh medium (15 mL final volume). After 24 h of culture, 12 mL of culture supernatant were again removed and replaced with fresh medium. On d 4, 6, and 8 of incubation, cultures were resuspended, and 200 ILL from each flask were dispensed into each of four wells of 96-well flat-bottomed plates. Proliferative levels in each culture were determined from these wells using 3H-thymidine as described above. In addition, 20 lzL were removed from each flask for analysis of cell numbers by Coulter counting.
Recovery of Phytohemagglutinin-lnduced Proliferation Postselenium Treatment Five 25-cm2 tissue-culture flasks were each inoculated with 2.5 mL of PBL at 2 x 106/mL in medium. Appropriate concentrations of 2 x concentrated selenium were then added in 2.5 mL of medium (5 mL final volume). After overnight incubation at 37~ cell suspensions were removed from flasks, washed 2• in medium to remove free Se, resuspended in 5 mL of medium, and incubated at 37~ for an additional 24 h. Following the second incubation, the cell suspensions were washed again, resuspended in 5 mL of medium, and 100 p,L of each culture were then dispensed into quadruplicate wells of 96-well plates. To each well, 100 FL of medium containing phytohemagglutinin (PHA, 2.0 ~g/mL, Miles Laboratories, Elkhart IN, USA) were added. Plates were incubated at 37~ for 3 d, and proliferation of cells was then measured using 3H-thymidine, as described above.
Cell Plumbers in Selenium-Treated ]vlLC One-way MLC were set up as described under mixed lymphocyte cultures (above). After 7 d of culture, 10 ~L of Hematall LA-Hgb lysing
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reagent (Fisher Scientific, Fairlawn NJ, USA) were added to each well. Twenty microliters from each well were then counted using a Coulter Counter (Coulter Electronics, Hialeah, FI, USA).
Normalization of 3H- Thymidine Incorporation Values The mean value in counts per minute (cpm) for 3H-thymidine incorporation by stimulated control cultures (no selenium) was calculated by pooling the control values for all individual donors (pooled control mean). A normalization factor was then calculated separately for each donor by dividing the pooled control mean by the control mean for that donor. Normalized values for each experimental set were calculated by multiplying the experimental mean of that set times the normalization factor for that donor.
RESOLTS Inhibition of Lymphocyte Proliferation in MLC The addition of selenium to lymphocytes stimulated to proliferate in one-way MLC resulted in a d o s e - d e p e n d e n t inhibition of proliferation (Fig. 1). The onset of inhibitory concentrations of selenium occurred in the range of 2-3 x 10- 4M, d e p e n d i n g on the time in culture. The statistical significance of inhibition by 2 • 10 ('M selenium was p < 0.001 on d 4 of culture, p < 0.1 on d 6, and not statistically significant on d 8, as analyzed by the Student's t-test for paired samples. Inhibition by selenium at 3 • 10 6M concentration and higher was statistically significant at the p < 0.001 level on all days of culture measured.
Inhibition of Lymphocyte Proliferation in Response to Tetanus Antigen When a d d e d to lymphocyte cultures, which had been stimulated with tetanus toxoid, 2 • 10 6M selenium showed highly significant levels of inhibition of proliferation (Fig. 2) on d 5 and 7 (p < 0.05) and less significant inhibition on d 9 (p < 0.1). Inhibition by 3 • 10 6M selenium was significant on all days tested (p < 0.02 on d 5 and 7 and p < 0.05 on d 9).
Recovery of Proliferative Capacity After Selenium Pretreatment The observed inhibition of lymphocyte proliferation by selenium could be interpreted as indicating that selenium at these concentrations was toxic to the responding lymphocytes; nonviable lymphocytes would be incapable of proliferation. Nonviability is defined in this case to mean a permanent and irreversible loss of proliferative capacity. In order to investigate this possibility, we incubated MLC for 8 h with a concentration Biolooical Trace Element Research
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0 0__a 2 9 DAY 4 9 DAY 6 i DAY 8 9x- U N S T I M L L ~ T E D
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[S4x lo-' M Fig. 1. Inhibition of MLC proliferation by selenium. When added to 1XMLC at the time of allogeneic stimulation, selenium caused the dose-dependent inhibition of proliferative response as measured by BH-thymidine incorporation. Inhibition was statistically significant in the range of 2-3 x 10
