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Correlation of early changes in amino acid transport and DNA synthesis in stimulated lymphocytes
CELLULAR
IMMUNOLOGY
Correlation
10,
319-323 (1974)
of Early Changes in Amino Acid Transport Synthesis in Stimulated Lymphocytes
and DNA
K. J. VAN DEN BERG AND I. BETEL Radiobiological
Institute
TNO,
Kleiweg 151, Rijswijk
Lange Received
August
(Z.H.),
The Netherlands
7. 1973
The early increase in the transport of aminoisobutyric acid (AIB) and DNA synthesis has been studied) in spleen cells from CBA mice, from congenitally athymic “nude” mice, and in rat thymocytes stimulated by Concanavalin A or lipopolysaccharide. A strict correlation is observed between the early increase in AIB transport and DNA synthesis in stimulated lymphocytes.
INTRODUCTION Binding of plant lectins to the surface of lymphoid cells induces a number of changes at the membrane level (1). Among the events occurring shortly after binding of the lectins are changes in surface charge (2)) increased transport of ions (3, 6), sugars (4, 7)) nucleosides (S), and amino acids (4, 9, 10). In addition, there are alterations in the activity of enzymes which are engaged in phospholipid synthesis (11, 12)) glycoprotein synthesis (13), ATP degradation (14)) and the synthesis of cyclic AMP (15). No information is available as to whether these early events are obligatory steps in the sequence of events which leads to blast formation and mitosis in the stimulated lymphocytes. The question has been raised by Stobo (16) whether lymphocytes which bind mitogen but are not induced to synthesize DNA, may undergo some of these early responses. In an attempt to answer this question we have studied the DNA synthesis and the early increase in amino acid transport in spleen cells of the CBA mouse, the congenitally athymic “nude” mouse and in rat thymocytes stimulated by Concanavalin A (Con A) and E. coli lipopolysaccharide (LPS). It has been demonstrated by Andersson et al. (17) that Con A and LPS are specific mitogens for the thymus derived (T) lymphocytes and bone marrow derived (B) lymphocytes, respectively. However, Con A and LPS bind equally well to T and B lymphocytes ( 18, 19). METHODS The spleens of 6-wk-old CBA mice and congenitally athymic “nude” mice and the thymi of 6-wk-old WAG/Rij rats were excised under sterile conditions. A lymphocyte suspension was made by gently pressing the finely cut spleen through a nylon gauze filter. The cells were collected and washed in HanksEagle’s medium with antibiotics. About 10’ nucleated cells/3 ml were cultured 31Y Copyri ht 0 All ru.%ts of
1974
by
Academic
Press,
Inc.
reproductionin any form reserved.
320
SHORT
COMMUNICATIONS
in this medium which was supplemented with 20% fetal calf serum. The lymphocytes were stimulated by addition of Con A (7 pg/ml) or LPS (10 rg/ml) to the cells in complete medium. Con A was obtained from CalBiochem (Los Angeles, CA) ; LPS (lipopolysaccharide from Escherichiu COZ 055: B5) from Difco (Detroit, MI). These concentrations of mitogens gave maximal DNA synthesis. At the times indicated, amino acid transport was measured; after washing and resuspending the cells in 0.5 ml Krebs buffer, 14C-aminoisobutyric acid (0.5 &i) was added to the cell suspension. After 60 min incubation at 37” amino acid uptake was determined by a filter method as previously described (20). DNA synthesis was determined after 48 hr of preincubation with mitogens by addition of 14C-thymidine (0.15 &i/22 ,ug) to 3 ml of the suspension in complete medium. The incubation was continued for 24 h at 37”. The cells were collected on glass fiber filters (GF/A, Whatman) and washed with 5 ml ice cold saline. The filters were transferred to a beaker with ice cold 10% TCA and left for at least 30 min at 0”. The precipitate was washed with 5 ml 5% TCA and finally with 1 ml ethanol: ether ( 1: 1). The filters were transferred to vials, dried and after addition of 15 ml scintillation fluid the radioactivity was determined. RESULTS
AND
DISCUSSION
The capacity to accumulate the nonmetabolizable amino acid aminoisobutyric acid (AIB) in a control CBA spleen suspension and one incubated with Con A is shown in Fig. 1 (A). There is an almost twofold increase in AIB uptake after
0 ‘p ’ E <
‘NUDE’
SPLEEN
CELLS
i
6
2
24
48
rwl TdR
FIG. 1. Effect of Con A on AIB transport and TdR incorporation in mouse spleen cells. Spleen cells from CBA mice (A) and from congenitally athymic “nude” mice (B) were preincubated with and without Con A. At the times indicated, AIB uptake was measured and after 48 hr of preincubation TdR incorporation was measured in the same suspension. Range bars represent 1 SEM.
SHORT
COMMUNICATIONS
,CBA SPLEEN
CELLS
321
0
control
I
tLPS
n
NUDE’
SPLEEN
CELLS
is
1
2
4
24
TdR
hours
FIG. 2. Effect of LPS on AIB uptake and TdR incorporation in mouse spleen cells. Spleen cells from CBA mice (A) and from congenitally athymic “nude” mice (B) were preincubated with and without LPS. At the times indicated, AIB uptake was measured and after 48 hr of preincubation TdR incorporaiton was measured in the same suspension. Range bars represent 1 SEM. 2 hr of incubation with Con A and a maximum level of uptake around 24 hr. This pattern is similar to the time course of AIB uptake obtained with rat lymph node cells (20). Increased thymidine incorporation was not observed before 24 hr of incubation and reached a maximum between 48 and 72 hr. The increase in AIB uptake thus precedes the increase in DNA synthesis. In a parallel study the same type of experiment was performed with a spleen suspension from the congenitally athymic “nude” mouse, which is deficient in T lymphocytes (21). Figure 1 (B) shows that Con A is not able to induce an early increase in AIB uptake and an increased DNA synthesis in these cells. Increasing the Con A concentration to 250 pg/ml induced only a marginal increase in AIB transport. CBA spleen cells incubated with LPS show an increase in AIB uptake as well as an increased DNi\ synthesis, although the increase in AIB uptake is delayed (Fig. 2 (A) ) . In the spleen cell suspension of the “nude” mouse, LPS also induces an increase in AIH uptake and an increase in DNA synthesis (Fig. 2(B) ) which is similar to that in the CBA spleen cell suspension. In the next experiment rat thymocytes were incubated with Con A or LPS and the AIB uptake and DNA synthesis were followed. In rat thymocytes, Con n induces an early increase in AIB uptake and later an increase in DNA synthesis, but LPS has no effect on AIB uptake or on thymidine incorporation (Fig. 3). Increasing the TAPS concentration to 250 pg/ml did not induce an early increase in RIB transport or DNA synthesis. It was noted that stimulation of DNA synthesis in rat thymocytes by Con A is of the same order of magnitude as the stimulation in spleen cells and peripheral lymphocytes.
322
SHORT
RI,
COMMUNICATIOXS
THYMOCYTES
2
4
24
TdR
FIG. 3. Effect of Con A and LPS on AIB uptake and TdR incorporation in rat thymocytes. WAG/Rij thymocytes were preincubated with and without Con -4 or LPS. At the times indicated AIB uptake was measured and after 48 hr of preincubation determined in the same suspension. Range bars represent 1 SEM.
TdR
incorporation
was
The present results demonstrate that, if DNA synthesis is induced by mitogens, it is preceded by an increase in AIB uptake. No early induction of increased AIB transport was observed when mitogens bind to lymphocytes but do not stimulate the lymphocytes to initiate DNA synthesis. The results indicate that the specificity of Con A and LPS to stimulate different cell populations is not restricted to DNA synthesis. The specificity seems already to be expressed as an early membrane event, including the increase in AIB transport. The increase in AIB transport can, therefore, be used as an early indication of lymphocyte stimulation. While these results demonstrate a correlation of the early increase in AIB transport and DNA synthesis in stimulated lymphocytes, they do not prove the existence of a causal relationship. It is also uncertain as to whether this correlation holds for other membrane phenomena; e.g., increased turnover of membrane phospholipids could only be observed in T cells stimulated by T cell mitogens and was not found in B cells (Betel and Van de Berg, in preparation). ACKNOWLEDGMENT WC thank
Mr.
J. B. de Vries
for Ilis skilled
technical
assistancr.
REFERENCES 1. 2. 3. 4. 5.
Greaves, M., amI Janossy, G., ?‘ran.@l. Rev. 11, 87, 1972. Currie, G. A., Nature’ 216, 694, 1967. Quastel, M. R., and Kaplan, J. G., Exp. Cell. RES. 63, 230, 1970. Averdunk, R., Hoppe-Seyler’s Z. Physiol. Ch.cm. 353, 79, 1972. Allwcod, G., Asherson, G. L., Davey, M. J., and Goodford, P. J., Immunology 1971. 6. Whitney, R. B., and Sutherland, R. M., Cell. Inwwnol. 5, 137, 1972. 7. Peters, J. H., and Hausen, P., Eur. J. Biochem. 19, 509, 1971.
21, SQ9,
SHORT
COMMUNICATIONS
323
8. Peters, J. H., and Hausen, P., Eur. J. Biochem. 19, 502, 1971. 9. Mendelsohn, J., Skinner, S. A., and Kornfeld, S., J. Cli%. Invest. 50, 818, 1971. 10. Van den Berg, K. J., and Betel, I., Exp. Cell Res. 66, 257, 1971. 11. Fisher, D. B., and Mueller, G. C., Biochenz. Biophys. Acta 248, 434, 1971. 12. Lucas, D. O., Shohet, S. B., and Merler, E., J. Zmmunol. 106, 768, 1971. 13. Hayden, G. A., Crowley, G. M., and Jameson, G .A., J. Biol. Chem. 245, 5827, 1970. 14. Novogrodsky, A., Biochem. Biophys. Acta 266, 343, 1972. 15. Smith, J. ‘W., Steiner, A. L., Newberry, Jr., W. M., and Parker, C. W., J. Cl&. Invest. 50, 60, 1972. 16. Stobo, J. D., Trarzspl. Rev. 11, 60, 1972. 17. Andersson, J., Mijller, G., and Sjiiberg, O., Cell. Immunol. 4, 381, 1972. 18. Stobo, J. D., Rosenthal, A. S., and Paul, W. E., J. Zmmunol. 108, 1, 1972. 19. Andersson, J., Sjijberg, O., and Miiller, G., Transpl. Rev. 11, 131, 1972. 20. Van den Berg, K. J., and Betel, I., Exp. Cell Res. 76, 63, 1973. 21. Raff, M. C., and Wortis, H. H., Zmmunology 18, 93, 1970.
IMMUNOLOGY
Correlation
10,
319-323 (1974)
of Early Changes in Amino Acid Transport Synthesis in Stimulated Lymphocytes
and DNA
K. J. VAN DEN BERG AND I. BETEL Radiobiological
Institute
TNO,
Kleiweg 151, Rijswijk
Lange Received
August
(Z.H.),
The Netherlands
7. 1973
The early increase in the transport of aminoisobutyric acid (AIB) and DNA synthesis has been studied) in spleen cells from CBA mice, from congenitally athymic “nude” mice, and in rat thymocytes stimulated by Concanavalin A or lipopolysaccharide. A strict correlation is observed between the early increase in AIB transport and DNA synthesis in stimulated lymphocytes.
INTRODUCTION Binding of plant lectins to the surface of lymphoid cells induces a number of changes at the membrane level (1). Among the events occurring shortly after binding of the lectins are changes in surface charge (2)) increased transport of ions (3, 6), sugars (4, 7)) nucleosides (S), and amino acids (4, 9, 10). In addition, there are alterations in the activity of enzymes which are engaged in phospholipid synthesis (11, 12)) glycoprotein synthesis (13), ATP degradation (14)) and the synthesis of cyclic AMP (15). No information is available as to whether these early events are obligatory steps in the sequence of events which leads to blast formation and mitosis in the stimulated lymphocytes. The question has been raised by Stobo (16) whether lymphocytes which bind mitogen but are not induced to synthesize DNA, may undergo some of these early responses. In an attempt to answer this question we have studied the DNA synthesis and the early increase in amino acid transport in spleen cells of the CBA mouse, the congenitally athymic “nude” mouse and in rat thymocytes stimulated by Concanavalin A (Con A) and E. coli lipopolysaccharide (LPS). It has been demonstrated by Andersson et al. (17) that Con A and LPS are specific mitogens for the thymus derived (T) lymphocytes and bone marrow derived (B) lymphocytes, respectively. However, Con A and LPS bind equally well to T and B lymphocytes ( 18, 19). METHODS The spleens of 6-wk-old CBA mice and congenitally athymic “nude” mice and the thymi of 6-wk-old WAG/Rij rats were excised under sterile conditions. A lymphocyte suspension was made by gently pressing the finely cut spleen through a nylon gauze filter. The cells were collected and washed in HanksEagle’s medium with antibiotics. About 10’ nucleated cells/3 ml were cultured 31Y Copyri ht 0 All ru.%ts of
1974
by
Academic
Press,
Inc.
reproductionin any form reserved.
320
SHORT
COMMUNICATIONS
in this medium which was supplemented with 20% fetal calf serum. The lymphocytes were stimulated by addition of Con A (7 pg/ml) or LPS (10 rg/ml) to the cells in complete medium. Con A was obtained from CalBiochem (Los Angeles, CA) ; LPS (lipopolysaccharide from Escherichiu COZ 055: B5) from Difco (Detroit, MI). These concentrations of mitogens gave maximal DNA synthesis. At the times indicated, amino acid transport was measured; after washing and resuspending the cells in 0.5 ml Krebs buffer, 14C-aminoisobutyric acid (0.5 &i) was added to the cell suspension. After 60 min incubation at 37” amino acid uptake was determined by a filter method as previously described (20). DNA synthesis was determined after 48 hr of preincubation with mitogens by addition of 14C-thymidine (0.15 &i/22 ,ug) to 3 ml of the suspension in complete medium. The incubation was continued for 24 h at 37”. The cells were collected on glass fiber filters (GF/A, Whatman) and washed with 5 ml ice cold saline. The filters were transferred to a beaker with ice cold 10% TCA and left for at least 30 min at 0”. The precipitate was washed with 5 ml 5% TCA and finally with 1 ml ethanol: ether ( 1: 1). The filters were transferred to vials, dried and after addition of 15 ml scintillation fluid the radioactivity was determined. RESULTS
AND
DISCUSSION
The capacity to accumulate the nonmetabolizable amino acid aminoisobutyric acid (AIB) in a control CBA spleen suspension and one incubated with Con A is shown in Fig. 1 (A). There is an almost twofold increase in AIB uptake after
0 ‘p ’ E <
‘NUDE’
SPLEEN
CELLS
i
6
2
24
48
rwl TdR
FIG. 1. Effect of Con A on AIB transport and TdR incorporation in mouse spleen cells. Spleen cells from CBA mice (A) and from congenitally athymic “nude” mice (B) were preincubated with and without Con A. At the times indicated, AIB uptake was measured and after 48 hr of preincubation TdR incorporation was measured in the same suspension. Range bars represent 1 SEM.
SHORT
COMMUNICATIONS
,CBA SPLEEN
CELLS
321
0
control
I
tLPS
n
NUDE’
SPLEEN
CELLS
is
1
2
4
24
TdR
hours
FIG. 2. Effect of LPS on AIB uptake and TdR incorporation in mouse spleen cells. Spleen cells from CBA mice (A) and from congenitally athymic “nude” mice (B) were preincubated with and without LPS. At the times indicated, AIB uptake was measured and after 48 hr of preincubation TdR incorporaiton was measured in the same suspension. Range bars represent 1 SEM. 2 hr of incubation with Con A and a maximum level of uptake around 24 hr. This pattern is similar to the time course of AIB uptake obtained with rat lymph node cells (20). Increased thymidine incorporation was not observed before 24 hr of incubation and reached a maximum between 48 and 72 hr. The increase in AIB uptake thus precedes the increase in DNA synthesis. In a parallel study the same type of experiment was performed with a spleen suspension from the congenitally athymic “nude” mouse, which is deficient in T lymphocytes (21). Figure 1 (B) shows that Con A is not able to induce an early increase in AIB uptake and an increased DNA synthesis in these cells. Increasing the Con A concentration to 250 pg/ml induced only a marginal increase in AIB transport. CBA spleen cells incubated with LPS show an increase in AIB uptake as well as an increased DNi\ synthesis, although the increase in AIB uptake is delayed (Fig. 2 (A) ) . In the spleen cell suspension of the “nude” mouse, LPS also induces an increase in AIH uptake and an increase in DNA synthesis (Fig. 2(B) ) which is similar to that in the CBA spleen cell suspension. In the next experiment rat thymocytes were incubated with Con A or LPS and the AIB uptake and DNA synthesis were followed. In rat thymocytes, Con n induces an early increase in AIB uptake and later an increase in DNA synthesis, but LPS has no effect on AIB uptake or on thymidine incorporation (Fig. 3). Increasing the TAPS concentration to 250 pg/ml did not induce an early increase in RIB transport or DNA synthesis. It was noted that stimulation of DNA synthesis in rat thymocytes by Con A is of the same order of magnitude as the stimulation in spleen cells and peripheral lymphocytes.
322
SHORT
RI,
COMMUNICATIOXS
THYMOCYTES
2
4
24
TdR
FIG. 3. Effect of Con A and LPS on AIB uptake and TdR incorporation in rat thymocytes. WAG/Rij thymocytes were preincubated with and without Con -4 or LPS. At the times indicated AIB uptake was measured and after 48 hr of preincubation determined in the same suspension. Range bars represent 1 SEM.
TdR
incorporation
was
The present results demonstrate that, if DNA synthesis is induced by mitogens, it is preceded by an increase in AIB uptake. No early induction of increased AIB transport was observed when mitogens bind to lymphocytes but do not stimulate the lymphocytes to initiate DNA synthesis. The results indicate that the specificity of Con A and LPS to stimulate different cell populations is not restricted to DNA synthesis. The specificity seems already to be expressed as an early membrane event, including the increase in AIB transport. The increase in AIB transport can, therefore, be used as an early indication of lymphocyte stimulation. While these results demonstrate a correlation of the early increase in AIB transport and DNA synthesis in stimulated lymphocytes, they do not prove the existence of a causal relationship. It is also uncertain as to whether this correlation holds for other membrane phenomena; e.g., increased turnover of membrane phospholipids could only be observed in T cells stimulated by T cell mitogens and was not found in B cells (Betel and Van de Berg, in preparation). ACKNOWLEDGMENT WC thank
Mr.
J. B. de Vries
for Ilis skilled
technical
assistancr.
REFERENCES 1. 2. 3. 4. 5.
Greaves, M., amI Janossy, G., ?‘ran.@l. Rev. 11, 87, 1972. Currie, G. A., Nature’ 216, 694, 1967. Quastel, M. R., and Kaplan, J. G., Exp. Cell. RES. 63, 230, 1970. Averdunk, R., Hoppe-Seyler’s Z. Physiol. Ch.cm. 353, 79, 1972. Allwcod, G., Asherson, G. L., Davey, M. J., and Goodford, P. J., Immunology 1971. 6. Whitney, R. B., and Sutherland, R. M., Cell. Inwwnol. 5, 137, 1972. 7. Peters, J. H., and Hausen, P., Eur. J. Biochem. 19, 509, 1971.
21, SQ9,
SHORT
COMMUNICATIONS
323
8. Peters, J. H., and Hausen, P., Eur. J. Biochem. 19, 502, 1971. 9. Mendelsohn, J., Skinner, S. A., and Kornfeld, S., J. Cli%. Invest. 50, 818, 1971. 10. Van den Berg, K. J., and Betel, I., Exp. Cell Res. 66, 257, 1971. 11. Fisher, D. B., and Mueller, G. C., Biochenz. Biophys. Acta 248, 434, 1971. 12. Lucas, D. O., Shohet, S. B., and Merler, E., J. Zmmunol. 106, 768, 1971. 13. Hayden, G. A., Crowley, G. M., and Jameson, G .A., J. Biol. Chem. 245, 5827, 1970. 14. Novogrodsky, A., Biochem. Biophys. Acta 266, 343, 1972. 15. Smith, J. ‘W., Steiner, A. L., Newberry, Jr., W. M., and Parker, C. W., J. Cl&. Invest. 50, 60, 1972. 16. Stobo, J. D., Trarzspl. Rev. 11, 60, 1972. 17. Andersson, J., Mijller, G., and Sjiiberg, O., Cell. Immunol. 4, 381, 1972. 18. Stobo, J. D., Rosenthal, A. S., and Paul, W. E., J. Zmmunol. 108, 1, 1972. 19. Andersson, J., Sjijberg, O., and Miiller, G., Transpl. Rev. 11, 131, 1972. 20. Van den Berg, K. J., and Betel, I., Exp. Cell Res. 76, 63, 1973. 21. Raff, M. C., and Wortis, H. H., Zmmunology 18, 93, 1970.