Int. Archs Allergy appl. Immun. 60: 89-96 (1979)
Serum Factors Affecting the Incorporation of |3H|thymidine by Lymphocytes Stimulated by Antigen IV. Comparison of Enhancement by Heated (56 °C) Serum and by 2-Mercaptoethanol
Donald R. Forsdyke Department of Biochemistry, Queen's University, Kingston, Ont.
Abstract. Serum preheated to 56 °C and 2-mercaptoethanol (ME) both enhance the stimulation (%>) by antigen of [3H]thymidine incorporation by lymph node cells from immunized rabbits. The enhancements show several similar features suggesting a common mechanism. Both enhancements show (i) non-specific components, affecting l:!H]thymidine incorporation in control and antigen-treated cultures proportionately, and (ii) antigenspecific components, having a greater proportionate effect on antigen-treated cultures than on control cultures. The non-specific components, in combination, are approximately addi tive. The specific components, in combination, are non-additive (mutually exclusive). With both preheated serum and ME the specific component of the enhancement is most evident late in the culture period and is greater at high antigen concentrations. The ME concentration needed for optimum enhancement is reduced if the serum concentration is reduced or if serum is preheated.
2-Mercaptoethanol (ME) enhances the response of cultured lymphoid cells to lec tins, antigens and homologous cells [1-4]. Several groups have found that serum fac tors are involved [5-9]. In a study of the re sponse to high concentrations of concanavalin A, we found enhancement both by ME and by serum heated to inactivate com plement. The enhancements were non-addi tive and showed several similar features, suggesting a common mechanism of action [10]. Heated serum also enhances the re sponse to antigen [11]. The purpose of the
present work was to compare the enhance ment of the response to antigen by heated serum with the enhancement by ME. If ME and heated serum were working through a common mechanism then, again, the en hancements might show similar characteris tics. Brief accounts of this work have been presented elsewhere [12, 13]. Materials and Methods Details of materials and methods were present ed previously [10, 11, 14]. ME was obtained from Eastman Kodak Co., Rochester, N.Y. Medium 199 was obtained from Grand Island Biological Co., N. Y. Giant keyhole limpet hemocyanin
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Introduction
Forsdyke
90
expressed as a percentage (I’) using the formula /’ 100 |(/1/C)-1| [cf. 14|. A represents the mean radioactivity of cultures containing a particular concentration of antigen and C represents the mean radioactivity of the corresponding control cultures without antigen. The significance of re sults was evaluated using Student’s t test.
Results
Disproportionate Enhancement by ME Late in the Culture Period. ME enhanced radioactive labelling with [3H]thymidine at all time-points studied, both in cultures with added KLH and in control cultures without added KLH. To determine the specificity for cultures with KLH [14], stimulations of labelling produced by KLH were expressed as a percentage of the labelling in the corre sponding control cultures without KLH (fig. 1). In the early stages of culture antigen dose-response curves in the presence and absence of ME diverged from each other only slightly (fig. la, b); at this time ME mainly enhanced labelling in control and antigen-treated cultures to a proportionate extent. Later in the culture period the do se-response curves diverged from each other more markedly (fig. lc, d). In all these fea tures, enhancement by ME resembled the enhancement produced by preheating the autologous serum used in cultures to 56 C for 20 min as was reported previously [ 111. Reduced Requirement for ME at Lower Serum Concentrations. Experiments of this series were carried out using a ’high’ KLH concentration (66 //g/ml) and serum which had not been preheated above 38 C. Fig ure 2 shows a representative experiment. With decreasing serum concentrations the ascending limbs of ME dose-response curves were shifted to the left and the ME
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(Kl.H) was obtained from Mann Research Labo ratories, New York, N.Y. [Me-3H] thymidine (2030Ci/mmol) was obtained from Amersham Corp., Oakville, Ont. Immunization. Female white rabbits (4-5 kg) were bled to obtain ‘preimmunization serum’ which was stored at -20 C. A few days later a toe-pad on each hind foot was injected with I mg of KLH. 2 weeks later, cell suspensions were prepared from popliteal lymph nodes for culture in vitro. In some experiments the immunization protocol was modified to increase the stimulation by heated se rum [cf. 13); only 0.1 mg of KL.H was injected and cells were prepared a month later. Cultures. Autologous preimmunization serum was heated at 56 C for 20 min in a water bath and then cooled by gently shaking in a water bath at room temperature. A serum so treated was re ferred to as ‘56 -serum’. Sera preheated at other temperatures were designated accordingly. Culture media were prewarmed at 38 C in air: C 0 2 (95:5). Cell suspensions were prepared by mincing lymph nodes for 3 min in medium 199 with finepointed scissors. The suspension was left 1 min for tissue fragments to settle and any fatty tissue to rise. The intermediate layer, containing free cells in suspension, was passed through a 80-mesh stainless steel filter and the cells were then pelletted by centrifugation (300 g, 3 min). The cells were resuspended in medium 199 and 1-ml vol umes were added to ‘master’ culture vessels (30 ml) which contained autologous serum and medium 199. After 5 min incubation at 38 C, ME was added and 1-ml volumes were immediate ly pipetted into glass round-bottomed culture tubes (1.4 cm internal diameter) containing var ious dilutions of KLH in medium 199 (50/ri). Each tube typically contained 2 X 106 nucleated cells, 25°/o autologous serum and 75°/o medium 199. In some experiments KLH was added directly to master culture vessels and the culture tubes contained various dilutions of ME. Radioactive Labelling. After 18-24 h of cul ture 1 ttC of [3H|thymidine was added in a vol ume of 20 «1. Cells were harvested by centrifuga tion (1,200 g, 4 min) at various time-points for the determination of acid-precipitable radioactivity with Hyamine as described previously [15]. To compensate for non-specific effects on radioactive labelling, stimulations of labelling by KLH were
Incorporation of | 3H]thymidine by Lymphocytes a
b
91
d
c
Concentration o f KLH, pg/ml
of acid-precipitable radioactivity after either (a) 28, (b) 45, (c) 53 or (d) 69 h. O = Cultures with out ME; A cultures with ME. Each point is the arithmetical mean of values from duplicate cul tures. In the case of points presented without the SEM the limits fell within, or close to, the area covered by the point. Values (c.p.m.) for control cultures (quadruplicates) without added KLH are shown in each figure.
Fig. 2. ME dose-response curves for cultures containing different concentrations of unheated auto logous serum. O 10% serum; A 1 15% serum; C = 20% serum; • 30% serum. 2 weeks after immunization (1 mg KLH/hind foot) popliteal lymph node cells were incubated (2.1 X 106,ml) with KLH (66 ug/ml) and various concentrations of autologous serum which had been collected prior to immunization. Various concentrations of ME were added at the initiation of culture and [3H]thymidine (1 «C; 20 Ci/mmol) at 21 h. Cells were harvested at 51 h. Each point is the arithmet ical mean value (c.p.m.) of triplicate culture + SEM.
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.
Fig. 1 Effects of ME on KLH dose-response curves at different times of culture. 2 weeks after immunization (1 mg KLH/hind foot), popliteal lymph node cells were incubated (2.1 X 106/ml) with varying concentrations of KLH in medium containing autologous serum (25%>) which had been collected prior to immunization. ME (100 uM) was added at the initiation of culture and (Me-3H]thymidine (1 «C; 20 Ci/mmol) after 22 h. Cells were harvested for the determination
92
Forsdyke b
Concentration o f ME, pM
Fig. 3. Effects of preheated autologous serum and ME, separately and in combination, on the radioactive labelling with [3H)thymidine of rabbit lymph node cells stimulated by KLH (40 rrg/ml). Figure 3a shows temperature-response curves for cultures containing various concentrations of ME. 0 = 0 uM\ • = 1 r-M ; A - 25 itM; A = 50 ftM; 100 nM; B 500 uM. In figure 3b the same data are plotted as ME dose-response curves for cultures containing serum preheated at different temperatures. O = 38 °C; • 45 C; A = 50 ’C;
A = 52 3C; □ - 56 C; ■ - 58 °C; V 62 °G; ▼ = 65 C. 2 weeks after immunization (1 mg KLH/hind foot), popliteal lymph node cells (2.4 X 106/ml) were incubated in medium contain ing various concentrations of ME and autologous serum (25Vo) which had been preheated for 20 min at various temperatures. [3H]thymidine (1 «C; 20 Ci/mmol) was added after 22 h of culture. Cells were harvested at 53 h. Each value shown is the mean of duplicate radioactivity values (c.p.m.). SEM are not shown.
concentration required for an optimum en hancement was reduced. A similar depend ence of the ME requirement on serum con centration was demonstrated in cultures stimulated by concanavalin A [10]. Additive Enhancements by Preheated Serum and ME in Cultures with Antigen: Reduced Requirement for ME in Preheated Serum. Figure 3 shows the effects of various combinations of ME and preheated serum on labelling in cultures stimulated by a high KLH concentration (40//g/ml). When the
results were plotted as a function of the temperature of preheating serum (fig. 3a). the temperature-response curves fell into three groups: (i) Zero and low ME concentrations: Curves for zero and 1 ,uM were similar. The curves showed most of the features de scribed previously [11], There was little change in radioactive labelling up to 50 C, then there was a steep increase in labelling over a relatively narrow range of tempera tures (50-53 C). At temperatures above
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 6:46:52 AM
Temperature, °C
Incorporation of | 3H]thymidine by Lymphocytes
58 C radioactive labelling plateaued or in creased less steeply. (ii) Intermediate ME concentrations: Curves for 25 and 50 fiM ME demonstrat ed synergistic effects between serum and ME. When added to cultures containing 38 C-serum, 25 uM ME produced no enhancement of labelling and 50 nM only a small enhancement. However, over the tem perature range 50-58 C there was a very steep increase of labelling to produce a final enhancement equal to the maximum ob tained at high ME concentrations at temper atures above 56 C. (Hi) High ME concentrations: Curves for 100 and 500 /iM ME were similar except for a relative inhibition in cultures contain ing 500 uM ME at 65 C (p < 0.002). In 38 C-serum labelling was enhanced by ME to approximately the same extent as was produced by 56 C-serum without ME. At each temperature the increased labelling in the presence of ME was approximately ad
93
ditive to the labelling in the absence of ME. Radioactive labelling increased steeply over the same narrow range of temperatures (5053 C) which produced the steep increase in labelling in cultures without ME. The curves then rose less steeply except for a small, but consistent (p < 0.05), depression of labelling at 58 °C. A possible explanation for the synergism at intermediate ME concentrations became evident when the same data were plotted as a function of ME concentration instead of temperature (fig. 3b). The dose-response curves showed that preheating serum shifted the ME concentration required to produce maximum labelling. Whereas in 38 C-se rum 100 uM ME was optimum, in 56 C-serum 50 aM ME was optimum and in 62 C-serum 25 /iM ME was optimum. The dose-response curves for 38, 45 and 50 C tended to be similar. With increasing temperatures above 50 °C the ascending limbs of ME dose-response curves became
Table I. Effects of preheated (56 C) serum and ME on labelling with [:iH]thymidine of rabbit lymph node cells cultured without added antigen1 Concentration of ME2 ¡jM
Radioactivity (c.p.m.)3 Period of labelling 18-24 h
38 38 56 56
0 100 0 50
661 ± 153 (0) 1,379 ± 89 (109) 1,329 ±75 (101) 1,876 ± 148 (184)
18-64 h 1.253 ± 1,673 ± 2,960 ± 4,216 ±
(0) 87 227 (34) 433 (136) 77 (236)
1 1 month after immunization (0.1 mg KLH/hind foot) popliteal lymph node cells (1.9 x lO'Vml) were cultured in medium containing autologous serum (23%). The scrum had been collected before immunization and was preheated at 56 or 38 C for 20 min shortly before initiating cultures. ME was added at the time of initiating cultures and [3H]thymidine (I /
Serum Factors Affecting the Incorporation of |3H|thymidine by Lymphocytes Stimulated by Antigen IV. Comparison of Enhancement by Heated (56 °C) Serum and by 2-Mercaptoethanol
Donald R. Forsdyke Department of Biochemistry, Queen's University, Kingston, Ont.
Abstract. Serum preheated to 56 °C and 2-mercaptoethanol (ME) both enhance the stimulation (%>) by antigen of [3H]thymidine incorporation by lymph node cells from immunized rabbits. The enhancements show several similar features suggesting a common mechanism. Both enhancements show (i) non-specific components, affecting l:!H]thymidine incorporation in control and antigen-treated cultures proportionately, and (ii) antigenspecific components, having a greater proportionate effect on antigen-treated cultures than on control cultures. The non-specific components, in combination, are approximately addi tive. The specific components, in combination, are non-additive (mutually exclusive). With both preheated serum and ME the specific component of the enhancement is most evident late in the culture period and is greater at high antigen concentrations. The ME concentration needed for optimum enhancement is reduced if the serum concentration is reduced or if serum is preheated.
2-Mercaptoethanol (ME) enhances the response of cultured lymphoid cells to lec tins, antigens and homologous cells [1-4]. Several groups have found that serum fac tors are involved [5-9]. In a study of the re sponse to high concentrations of concanavalin A, we found enhancement both by ME and by serum heated to inactivate com plement. The enhancements were non-addi tive and showed several similar features, suggesting a common mechanism of action [10]. Heated serum also enhances the re sponse to antigen [11]. The purpose of the
present work was to compare the enhance ment of the response to antigen by heated serum with the enhancement by ME. If ME and heated serum were working through a common mechanism then, again, the en hancements might show similar characteris tics. Brief accounts of this work have been presented elsewhere [12, 13]. Materials and Methods Details of materials and methods were present ed previously [10, 11, 14]. ME was obtained from Eastman Kodak Co., Rochester, N.Y. Medium 199 was obtained from Grand Island Biological Co., N. Y. Giant keyhole limpet hemocyanin
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 6:46:52 AM
Introduction
Forsdyke
90
expressed as a percentage (I’) using the formula /’ 100 |(/1/C)-1| [cf. 14|. A represents the mean radioactivity of cultures containing a particular concentration of antigen and C represents the mean radioactivity of the corresponding control cultures without antigen. The significance of re sults was evaluated using Student’s t test.
Results
Disproportionate Enhancement by ME Late in the Culture Period. ME enhanced radioactive labelling with [3H]thymidine at all time-points studied, both in cultures with added KLH and in control cultures without added KLH. To determine the specificity for cultures with KLH [14], stimulations of labelling produced by KLH were expressed as a percentage of the labelling in the corre sponding control cultures without KLH (fig. 1). In the early stages of culture antigen dose-response curves in the presence and absence of ME diverged from each other only slightly (fig. la, b); at this time ME mainly enhanced labelling in control and antigen-treated cultures to a proportionate extent. Later in the culture period the do se-response curves diverged from each other more markedly (fig. lc, d). In all these fea tures, enhancement by ME resembled the enhancement produced by preheating the autologous serum used in cultures to 56 C for 20 min as was reported previously [ 111. Reduced Requirement for ME at Lower Serum Concentrations. Experiments of this series were carried out using a ’high’ KLH concentration (66 //g/ml) and serum which had not been preheated above 38 C. Fig ure 2 shows a representative experiment. With decreasing serum concentrations the ascending limbs of ME dose-response curves were shifted to the left and the ME
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 6:46:52 AM
(Kl.H) was obtained from Mann Research Labo ratories, New York, N.Y. [Me-3H] thymidine (2030Ci/mmol) was obtained from Amersham Corp., Oakville, Ont. Immunization. Female white rabbits (4-5 kg) were bled to obtain ‘preimmunization serum’ which was stored at -20 C. A few days later a toe-pad on each hind foot was injected with I mg of KLH. 2 weeks later, cell suspensions were prepared from popliteal lymph nodes for culture in vitro. In some experiments the immunization protocol was modified to increase the stimulation by heated se rum [cf. 13); only 0.1 mg of KL.H was injected and cells were prepared a month later. Cultures. Autologous preimmunization serum was heated at 56 C for 20 min in a water bath and then cooled by gently shaking in a water bath at room temperature. A serum so treated was re ferred to as ‘56 -serum’. Sera preheated at other temperatures were designated accordingly. Culture media were prewarmed at 38 C in air: C 0 2 (95:5). Cell suspensions were prepared by mincing lymph nodes for 3 min in medium 199 with finepointed scissors. The suspension was left 1 min for tissue fragments to settle and any fatty tissue to rise. The intermediate layer, containing free cells in suspension, was passed through a 80-mesh stainless steel filter and the cells were then pelletted by centrifugation (300 g, 3 min). The cells were resuspended in medium 199 and 1-ml vol umes were added to ‘master’ culture vessels (30 ml) which contained autologous serum and medium 199. After 5 min incubation at 38 C, ME was added and 1-ml volumes were immediate ly pipetted into glass round-bottomed culture tubes (1.4 cm internal diameter) containing var ious dilutions of KLH in medium 199 (50/ri). Each tube typically contained 2 X 106 nucleated cells, 25°/o autologous serum and 75°/o medium 199. In some experiments KLH was added directly to master culture vessels and the culture tubes contained various dilutions of ME. Radioactive Labelling. After 18-24 h of cul ture 1 ttC of [3H|thymidine was added in a vol ume of 20 «1. Cells were harvested by centrifuga tion (1,200 g, 4 min) at various time-points for the determination of acid-precipitable radioactivity with Hyamine as described previously [15]. To compensate for non-specific effects on radioactive labelling, stimulations of labelling by KLH were
Incorporation of | 3H]thymidine by Lymphocytes a
b
91
d
c
Concentration o f KLH, pg/ml
of acid-precipitable radioactivity after either (a) 28, (b) 45, (c) 53 or (d) 69 h. O = Cultures with out ME; A cultures with ME. Each point is the arithmetical mean of values from duplicate cul tures. In the case of points presented without the SEM the limits fell within, or close to, the area covered by the point. Values (c.p.m.) for control cultures (quadruplicates) without added KLH are shown in each figure.
Fig. 2. ME dose-response curves for cultures containing different concentrations of unheated auto logous serum. O 10% serum; A 1 15% serum; C = 20% serum; • 30% serum. 2 weeks after immunization (1 mg KLH/hind foot) popliteal lymph node cells were incubated (2.1 X 106,ml) with KLH (66 ug/ml) and various concentrations of autologous serum which had been collected prior to immunization. Various concentrations of ME were added at the initiation of culture and [3H]thymidine (1 «C; 20 Ci/mmol) at 21 h. Cells were harvested at 51 h. Each point is the arithmet ical mean value (c.p.m.) of triplicate culture + SEM.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 6:46:52 AM
.
Fig. 1 Effects of ME on KLH dose-response curves at different times of culture. 2 weeks after immunization (1 mg KLH/hind foot), popliteal lymph node cells were incubated (2.1 X 106/ml) with varying concentrations of KLH in medium containing autologous serum (25%>) which had been collected prior to immunization. ME (100 uM) was added at the initiation of culture and (Me-3H]thymidine (1 «C; 20 Ci/mmol) after 22 h. Cells were harvested for the determination
92
Forsdyke b
Concentration o f ME, pM
Fig. 3. Effects of preheated autologous serum and ME, separately and in combination, on the radioactive labelling with [3H)thymidine of rabbit lymph node cells stimulated by KLH (40 rrg/ml). Figure 3a shows temperature-response curves for cultures containing various concentrations of ME. 0 = 0 uM\ • = 1 r-M ; A - 25 itM; A = 50 ftM; 100 nM; B 500 uM. In figure 3b the same data are plotted as ME dose-response curves for cultures containing serum preheated at different temperatures. O = 38 °C; • 45 C; A = 50 ’C;
A = 52 3C; □ - 56 C; ■ - 58 °C; V 62 °G; ▼ = 65 C. 2 weeks after immunization (1 mg KLH/hind foot), popliteal lymph node cells (2.4 X 106/ml) were incubated in medium contain ing various concentrations of ME and autologous serum (25Vo) which had been preheated for 20 min at various temperatures. [3H]thymidine (1 «C; 20 Ci/mmol) was added after 22 h of culture. Cells were harvested at 53 h. Each value shown is the mean of duplicate radioactivity values (c.p.m.). SEM are not shown.
concentration required for an optimum en hancement was reduced. A similar depend ence of the ME requirement on serum con centration was demonstrated in cultures stimulated by concanavalin A [10]. Additive Enhancements by Preheated Serum and ME in Cultures with Antigen: Reduced Requirement for ME in Preheated Serum. Figure 3 shows the effects of various combinations of ME and preheated serum on labelling in cultures stimulated by a high KLH concentration (40//g/ml). When the
results were plotted as a function of the temperature of preheating serum (fig. 3a). the temperature-response curves fell into three groups: (i) Zero and low ME concentrations: Curves for zero and 1 ,uM were similar. The curves showed most of the features de scribed previously [11], There was little change in radioactive labelling up to 50 C, then there was a steep increase in labelling over a relatively narrow range of tempera tures (50-53 C). At temperatures above
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/8/2018 6:46:52 AM
Temperature, °C
Incorporation of | 3H]thymidine by Lymphocytes
58 C radioactive labelling plateaued or in creased less steeply. (ii) Intermediate ME concentrations: Curves for 25 and 50 fiM ME demonstrat ed synergistic effects between serum and ME. When added to cultures containing 38 C-serum, 25 uM ME produced no enhancement of labelling and 50 nM only a small enhancement. However, over the tem perature range 50-58 C there was a very steep increase of labelling to produce a final enhancement equal to the maximum ob tained at high ME concentrations at temper atures above 56 C. (Hi) High ME concentrations: Curves for 100 and 500 /iM ME were similar except for a relative inhibition in cultures contain ing 500 uM ME at 65 C (p < 0.002). In 38 C-serum labelling was enhanced by ME to approximately the same extent as was produced by 56 C-serum without ME. At each temperature the increased labelling in the presence of ME was approximately ad
93
ditive to the labelling in the absence of ME. Radioactive labelling increased steeply over the same narrow range of temperatures (5053 C) which produced the steep increase in labelling in cultures without ME. The curves then rose less steeply except for a small, but consistent (p < 0.05), depression of labelling at 58 °C. A possible explanation for the synergism at intermediate ME concentrations became evident when the same data were plotted as a function of ME concentration instead of temperature (fig. 3b). The dose-response curves showed that preheating serum shifted the ME concentration required to produce maximum labelling. Whereas in 38 C-se rum 100 uM ME was optimum, in 56 C-serum 50 aM ME was optimum and in 62 C-serum 25 /iM ME was optimum. The dose-response curves for 38, 45 and 50 C tended to be similar. With increasing temperatures above 50 °C the ascending limbs of ME dose-response curves became
Table I. Effects of preheated (56 C) serum and ME on labelling with [:iH]thymidine of rabbit lymph node cells cultured without added antigen1 Concentration of ME2 ¡jM
Radioactivity (c.p.m.)3 Period of labelling 18-24 h
38 38 56 56
0 100 0 50
661 ± 153 (0) 1,379 ± 89 (109) 1,329 ±75 (101) 1,876 ± 148 (184)
18-64 h 1.253 ± 1,673 ± 2,960 ± 4,216 ±
(0) 87 227 (34) 433 (136) 77 (236)
1 1 month after immunization (0.1 mg KLH/hind foot) popliteal lymph node cells (1.9 x lO'Vml) were cultured in medium containing autologous serum (23%). The scrum had been collected before immunization and was preheated at 56 or 38 C for 20 min shortly before initiating cultures. ME was added at the time of initiating cultures and [3H]thymidine (I /
