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Differential effect of medium change in lymphocyte cultures stimulated by PPD tuberculin or PHA
CELLULAR
3,
IMMUSOLOCY
Differential
333-337(1972)
Effect of Medium Change in Lymphocyte Stimulated by PPD Tuberculin or PHA BENGT S.
Departwr~~t
Cultures
NILSSON
of Tlzovocic Medicinr, Kovolirzska Sjukhrset. Ser-aFrrlerlasa.rettet, Stockl7olnt,
and Tva~~spla~~tation Laboratory, Sevede+a
‘1 he aim of this work was to study whether soluble factors produced in stimulated lymphocyte cultures play any role in the events in the cultures where they have been produced. When the whole culture medium in PPDstimulated lymphocyte cultures was removed, either daily or on day 3, and replaced hy fresh medium with the same antigen concentration, a marked decrease in thymidine uptake resulted. The same operation in PH-4-stimulated cultures resulted in an increased thymidine uptake. Suitable controls showed that these effects were not due to artifacts, caused by manipulation of the cultures. If the old medium was replaced hy medium from other stimulated lymphocyte cultures, the response was restored to normal levels. Medium from unstimulated cultures had a slight effect, which was considerably increased by addition of PPD. The findings may he related to unspecific conditioning factors. but possibly also to specifically lymphocyte-activating material.
INTRODUCTION Supernatant fractiolx in normal or stimulated lymphocyte cultures have been shown to contain material which can activate other lymphocytes in various ways (I-9). Investigators dealing with this subject have usually removed supernatant fluids from one set of lymphocyte cultures and studied their effects on a second set of allogeneic or autochtonous cultures. The aim of the present study was to investigate whether soluble materials produced in a particular culture have any importance for the events occurring in this culture. This problem was approached by removing the whole culture medium in stimulated lymphocyte cultures at different times during the culture period and replacing it with fresh medium with the same antigen concentration or with medium from other lymphocyte cultures treated in various ways. Comparison of cell proliferation in cultures was performed by measurements of thymidine uptake.
MATERIALS AND METHODS
Heparitlized blood from healthy, tuberculin-reactive blood donors was used throughout. The procedures for separation of blood and cell culture have been described in detail elsewhere (10). In short, cultures consisted of 10” lymphocytes in 1 ml of Eagle’s suspension medium, supplemented with 10% human AB-serum. PPD-tuberculin (Statens Seruminstitut, Copenhagen j was used in final concentration 10 pg/ml. Adsorption of PPD to bentonite was performed essentially as
333 0
1972
by
Academic
Press,
Inc.
334
NILSSON
described by Cheng and Talmage ( 11 ). This bentonite-adsorbed PPD is strong]? stimulatory to lymphocytes, often more stimulatory than soluble PPD in the same concentration (data to be published). Phytohaemagglutinin, PH:1, (\il’ellcome Foundation, England) was used in final dilution 1p2. Medium change was performed either daily or on day 3, as stated in Results. The cells in culture were centrifuged 5 min at 4009 and the whole supernatant carefully sucked off with syringe and needle without disturbing the cells. The same volume of medium was added immediately, either fresh medium with the same antigen concentration, or medium from other lymphocyte cultures. Medium that was transferred to other cultures was either centrifuged or Millipore-filtered (pore size 0.45 p) before use. The control cultures, in which no medium change was made, were centrifuged in the same way as described above. DNA synthesis was measured on day 5-6 by measurement of 24-hr uptake of l*C-thymidine. Results were expressed as cpm/culture (mean value for three cultures minus background activity I+ SE). Some results were expressed as percentage of change of thymidine uptake in comparison with control cultures. RESULTS When the whole culture medium in PPD-stimulated cultures was removed daily and replaced by fresh medium with the same antigen concentration, the thymidine uptake was greatly decreased (Fig. 1). In PHA cultures, the same operation resulted in an increased uptake (Fig. 2). The mean suppression of thymidine uptake in the PPD cultures was 83 t 6% and in the PHA cultures the mean increase was 31 2 770 (mean values from 10 experiments). If fresh antigen leas added, without
FIG. 1. Effect of daily renewal of medium in PPD-stimulated lymphocyte cultures. Each point represents the mean value of three cultures *SE and each line represents one experiment. For details, see text.
EFFECT
OF
MEDIUM
CHANGE
IN
LYMPHOCYTE
CULTURES
335
2000 i cpm
"0
medium
dally
medum
ctmge ciwlge FIG. 2. Effect of daily renelval of medium in PHA-stimulated lymphocyte cultures. removal of the old medium, a normal response was found. The same was true when cent,rifugated or when Millipore-filtered old medium was returned to the same cultures (this has been tested in 15 experiments, always with the same result). If only part of the medium was changed, there was an unchanged or slightly increased thymidine uptake. In the above experiments, the medium replacements involved addition of fresh antigen (and possibly removal of in some way modified antigen). In other experiments anti,gen was used in a particulate form, such as PPD adsorbed to bentonite or HL-A antigens on lymphoid cells (mixed lymphocyte culture, MLC). At the medium change in these cultures, the antigen was centrifuged down together with the cells, and consequently the old medium was replaced by fresh medium without antigen. With bentonite-PPD, the results were the same as with soluble PPD mean percentage of reduction of thymidine uptake 90 + 1% in three experiments). In the MLC the reduction was less marked (51 -t 11% in five experiments). If the medium change was performed only once, on day 3, instead of each day during the culture period, the reduction of thymidine uptake was less marked, but still strongly significant. In order to simplify the experiments and minimize manipulations with the cultures, the subsequent experiments were performed with medium change only on day 3. As mentioned above, a normal response was found if the medium was returned to the cultures after it had been centrifuged or Millipore-filtered. A normal response was also found in most cases if the old medium was replaced by medium from PPD-stimulated cultures, derived from other cell donors (Table 1, col. d). Medium from cells cultured without antigen, or with antigen to which they did not resp0n.d (such as tetanus toxoid) was in some experiments without effect, in
336
NILSSON TABLE
EFFECT
I
THE OLD MEDIUV WITH MEDIUM FROM UNSTIMULATED OR PPDOF I~I~PIACING CULT~KES FROM OTHER CELL DONORS. MEDIUM CHASGE &'AS PERFORMED ON DAY 3
STIMI-LATIW
‘% Reduction
of thl-midine uptake in comparison with cultures no medium change was made Old medium replaced b>
il
b
Fresh medium with antigen
~ledium from unstim. cult.
6.3
62
71 90 68 71 72.6
58 62 64 65 62.2
c Medium from unstim. cult. + PPD 10 ~*g/rnl 41 59 40 53 41 46.8
in which
d Medium from PPD-stim. cult. 0 40 40 9 +47 8.4
others it had a slight effect (Table 1, col. b ). Medium from unstimulated cultures, with addition of J’l’D, gave an increased response, which, however, did not reach the levels fomid with medium from PPD-stimulated cultures (Table 1. col. c). DISCUSSION The above finding-s indicate that soluble factors, formed by antigen-stimulated lymphocytes, arc of great importance for further proliferation and division of the cells. It seems improbable that the sharp decrease in thymidine uptake after medium change in Pl’D cultures could be an artifact, caused by manipulation of the cultures, first, because the effect in PHA cultures was the opposite, second, because the response could be restored by Millipore-filtered or centrifuged old medium. The question whether the effect is due to specific, lymphocyte-activating sub stances or merely to unspecific “conditioning” factors is not resolved by the present results. However, the finding that the PPD reactivity, in which the need for recruitment of nonimmune cells is greatest, was most affected. whereas the response to histocompatibility ant&ens, in which the number of initially responding cells is considerably higher, was more moderately affected, suggests that the effects ;tre at least partly due to some type of specifically lymphocyte-activatingmaterial. If this is so, the experimental system used here may offer new possibilities to study this complex subject. ACKNOWLEDGMENTS I thank Dr. Giiran MGller for valuable advice during the experiments and help in preparation of the manuscript. Miss Dana iistrijm has given expert technical assistance, which is gratefully acknowledged. This study was supported by grants from the Swedish National Association against Heart and Lung Diseases, Carin Tryggers Minnesfond, the Swedish Medical Research Council. The author is receiving a personal grant from Lisa and Johan Grijnbergs Stiftelse. Part of this work was also supported by a personal grant from The Swedish National Society against Cancer.
EFFECT
OF
MEDIUM
CHANGE
IN
LYMPHOCYTE
CULTITRES
337
REFERENCES 1. 2. 3. 4. 5. 6. 7. S. 9. 10. 11.
Kasakura, S., and Lowenstein, L.. h’nfwe London 208, 794. 1965. Gordon. J,. and MacLean, L. D., X:crturr Lorzdorc 208, 795, 1965. Kasakura, S.. and Lowenstein, I,., Trmsplnlzfnfion 5, 459. 1967. Valentine, F. T., and Lawrence, H. S., Scie~e 165, 1014, 1969. Maini, I?. X., Bryceson, A. D. RI., Wolstencroft, K. A., and Dumonde. D. C.. Xatztve Loitdo~t 224. 43, 1969. Dum~~ntle. D.C.. \Volstencroft, K. 4., Panayi, G. S., Matthe\q M., Morley. J., and Howson, LT. T.. A\rnfwe Lotzdort 224, 38, 1969. Kasakul-a. S., J. Iv~mxol. 105, 1162, 1970. Janis. M., and Bach, F. T.. Nature Lomfoa 225, 238, 1970. Powles. K., Balchin, L., Curie, G. A., and Alexander, P., AYufwc Londm 231, 161, 1971. Nilsson, B. S.. Cell. Z~zumwol. In press. Cheng, IV.‘. C.. and Talmage. D. W., .I. I~u~tol. 103, 1385, 1969.
3,
IMMUSOLOCY
Differential
333-337(1972)
Effect of Medium Change in Lymphocyte Stimulated by PPD Tuberculin or PHA BENGT S.
Departwr~~t
Cultures
NILSSON
of Tlzovocic Medicinr, Kovolirzska Sjukhrset. Ser-aFrrlerlasa.rettet, Stockl7olnt,
and Tva~~spla~~tation Laboratory, Sevede+a
‘1 he aim of this work was to study whether soluble factors produced in stimulated lymphocyte cultures play any role in the events in the cultures where they have been produced. When the whole culture medium in PPDstimulated lymphocyte cultures was removed, either daily or on day 3, and replaced hy fresh medium with the same antigen concentration, a marked decrease in thymidine uptake resulted. The same operation in PH-4-stimulated cultures resulted in an increased thymidine uptake. Suitable controls showed that these effects were not due to artifacts, caused by manipulation of the cultures. If the old medium was replaced hy medium from other stimulated lymphocyte cultures, the response was restored to normal levels. Medium from unstimulated cultures had a slight effect, which was considerably increased by addition of PPD. The findings may he related to unspecific conditioning factors. but possibly also to specifically lymphocyte-activating material.
INTRODUCTION Supernatant fractiolx in normal or stimulated lymphocyte cultures have been shown to contain material which can activate other lymphocytes in various ways (I-9). Investigators dealing with this subject have usually removed supernatant fluids from one set of lymphocyte cultures and studied their effects on a second set of allogeneic or autochtonous cultures. The aim of the present study was to investigate whether soluble materials produced in a particular culture have any importance for the events occurring in this culture. This problem was approached by removing the whole culture medium in stimulated lymphocyte cultures at different times during the culture period and replacing it with fresh medium with the same antigen concentration or with medium from other lymphocyte cultures treated in various ways. Comparison of cell proliferation in cultures was performed by measurements of thymidine uptake.
MATERIALS AND METHODS
Heparitlized blood from healthy, tuberculin-reactive blood donors was used throughout. The procedures for separation of blood and cell culture have been described in detail elsewhere (10). In short, cultures consisted of 10” lymphocytes in 1 ml of Eagle’s suspension medium, supplemented with 10% human AB-serum. PPD-tuberculin (Statens Seruminstitut, Copenhagen j was used in final concentration 10 pg/ml. Adsorption of PPD to bentonite was performed essentially as
333 0
1972
by
Academic
Press,
Inc.
334
NILSSON
described by Cheng and Talmage ( 11 ). This bentonite-adsorbed PPD is strong]? stimulatory to lymphocytes, often more stimulatory than soluble PPD in the same concentration (data to be published). Phytohaemagglutinin, PH:1, (\il’ellcome Foundation, England) was used in final dilution 1p2. Medium change was performed either daily or on day 3, as stated in Results. The cells in culture were centrifuged 5 min at 4009 and the whole supernatant carefully sucked off with syringe and needle without disturbing the cells. The same volume of medium was added immediately, either fresh medium with the same antigen concentration, or medium from other lymphocyte cultures. Medium that was transferred to other cultures was either centrifuged or Millipore-filtered (pore size 0.45 p) before use. The control cultures, in which no medium change was made, were centrifuged in the same way as described above. DNA synthesis was measured on day 5-6 by measurement of 24-hr uptake of l*C-thymidine. Results were expressed as cpm/culture (mean value for three cultures minus background activity I+ SE). Some results were expressed as percentage of change of thymidine uptake in comparison with control cultures. RESULTS When the whole culture medium in PPD-stimulated cultures was removed daily and replaced by fresh medium with the same antigen concentration, the thymidine uptake was greatly decreased (Fig. 1). In PHA cultures, the same operation resulted in an increased uptake (Fig. 2). The mean suppression of thymidine uptake in the PPD cultures was 83 t 6% and in the PHA cultures the mean increase was 31 2 770 (mean values from 10 experiments). If fresh antigen leas added, without
FIG. 1. Effect of daily renewal of medium in PPD-stimulated lymphocyte cultures. Each point represents the mean value of three cultures *SE and each line represents one experiment. For details, see text.
EFFECT
OF
MEDIUM
CHANGE
IN
LYMPHOCYTE
CULTURES
335
2000 i cpm
"0
medium
dally
medum
ctmge ciwlge FIG. 2. Effect of daily renelval of medium in PHA-stimulated lymphocyte cultures. removal of the old medium, a normal response was found. The same was true when cent,rifugated or when Millipore-filtered old medium was returned to the same cultures (this has been tested in 15 experiments, always with the same result). If only part of the medium was changed, there was an unchanged or slightly increased thymidine uptake. In the above experiments, the medium replacements involved addition of fresh antigen (and possibly removal of in some way modified antigen). In other experiments anti,gen was used in a particulate form, such as PPD adsorbed to bentonite or HL-A antigens on lymphoid cells (mixed lymphocyte culture, MLC). At the medium change in these cultures, the antigen was centrifuged down together with the cells, and consequently the old medium was replaced by fresh medium without antigen. With bentonite-PPD, the results were the same as with soluble PPD mean percentage of reduction of thymidine uptake 90 + 1% in three experiments). In the MLC the reduction was less marked (51 -t 11% in five experiments). If the medium change was performed only once, on day 3, instead of each day during the culture period, the reduction of thymidine uptake was less marked, but still strongly significant. In order to simplify the experiments and minimize manipulations with the cultures, the subsequent experiments were performed with medium change only on day 3. As mentioned above, a normal response was found if the medium was returned to the cultures after it had been centrifuged or Millipore-filtered. A normal response was also found in most cases if the old medium was replaced by medium from PPD-stimulated cultures, derived from other cell donors (Table 1, col. d). Medium from cells cultured without antigen, or with antigen to which they did not resp0n.d (such as tetanus toxoid) was in some experiments without effect, in
336
NILSSON TABLE
EFFECT
I
THE OLD MEDIUV WITH MEDIUM FROM UNSTIMULATED OR PPDOF I~I~PIACING CULT~KES FROM OTHER CELL DONORS. MEDIUM CHASGE &'AS PERFORMED ON DAY 3
STIMI-LATIW
‘% Reduction
of thl-midine uptake in comparison with cultures no medium change was made Old medium replaced b>
il
b
Fresh medium with antigen
~ledium from unstim. cult.
6.3
62
71 90 68 71 72.6
58 62 64 65 62.2
c Medium from unstim. cult. + PPD 10 ~*g/rnl 41 59 40 53 41 46.8
in which
d Medium from PPD-stim. cult. 0 40 40 9 +47 8.4
others it had a slight effect (Table 1, col. b ). Medium from unstimulated cultures, with addition of J’l’D, gave an increased response, which, however, did not reach the levels fomid with medium from PPD-stimulated cultures (Table 1. col. c). DISCUSSION The above finding-s indicate that soluble factors, formed by antigen-stimulated lymphocytes, arc of great importance for further proliferation and division of the cells. It seems improbable that the sharp decrease in thymidine uptake after medium change in Pl’D cultures could be an artifact, caused by manipulation of the cultures, first, because the effect in PHA cultures was the opposite, second, because the response could be restored by Millipore-filtered or centrifuged old medium. The question whether the effect is due to specific, lymphocyte-activating sub stances or merely to unspecific “conditioning” factors is not resolved by the present results. However, the finding that the PPD reactivity, in which the need for recruitment of nonimmune cells is greatest, was most affected. whereas the response to histocompatibility ant&ens, in which the number of initially responding cells is considerably higher, was more moderately affected, suggests that the effects ;tre at least partly due to some type of specifically lymphocyte-activatingmaterial. If this is so, the experimental system used here may offer new possibilities to study this complex subject. ACKNOWLEDGMENTS I thank Dr. Giiran MGller for valuable advice during the experiments and help in preparation of the manuscript. Miss Dana iistrijm has given expert technical assistance, which is gratefully acknowledged. This study was supported by grants from the Swedish National Association against Heart and Lung Diseases, Carin Tryggers Minnesfond, the Swedish Medical Research Council. The author is receiving a personal grant from Lisa and Johan Grijnbergs Stiftelse. Part of this work was also supported by a personal grant from The Swedish National Society against Cancer.
EFFECT
OF
MEDIUM
CHANGE
IN
LYMPHOCYTE
CULTITRES
337
REFERENCES 1. 2. 3. 4. 5. 6. 7. S. 9. 10. 11.
Kasakura, S., and Lowenstein, L.. h’nfwe London 208, 794. 1965. Gordon. J,. and MacLean, L. D., X:crturr Lorzdorc 208, 795, 1965. Kasakura, S.. and Lowenstein, I,., Trmsplnlzfnfion 5, 459. 1967. Valentine, F. T., and Lawrence, H. S., Scie~e 165, 1014, 1969. Maini, I?. X., Bryceson, A. D. RI., Wolstencroft, K. A., and Dumonde. D. C.. Xatztve Loitdo~t 224. 43, 1969. Dum~~ntle. D.C.. \Volstencroft, K. 4., Panayi, G. S., Matthe\q M., Morley. J., and Howson, LT. T.. A\rnfwe Lotzdort 224, 38, 1969. Kasakul-a. S., J. Iv~mxol. 105, 1162, 1970. Janis. M., and Bach, F. T.. Nature Lomfoa 225, 238, 1970. Powles. K., Balchin, L., Curie, G. A., and Alexander, P., AYufwc Londm 231, 161, 1971. Nilsson, B. S.. Cell. Z~zumwol. In press. Cheng, IV.‘. C.. and Talmage. D. W., .I. I~u~tol. 103, 1385, 1969.