576th MEETING, LONDON
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stimulation bymitogen (Table 1). As little as 1OpM-adenosine inhibited thestimulation of uridine uptake by mitogen by 79% while decreasing the rate of uptake by unstimulated lymphocytes by only 24 %. Adenine or ATP at concentrations up to 1mM had much less effect on uridine uptake by lymphocytes incubated with or without mitogen. The addition of phytohaemagglutinin to lymphocytes also leads to a large but transient increase in intracellular prostaglandin Ez within the first hour, which can be prevented by prior addition of the prostaglandin synthesis inhibitors indomethacin or phenylbutazone (C. A. Phillips & J. E. Kay, unpublished work). However, Fig. 1 shows that concentrations of indomethacin or phenylbutazone that completely suppress the usual transient increase d o not affect the early increase in t3H]uridine uptake due to phytohaemagglutinin. These studies suggest that the rapid increase in [3H]uridine uptake after addition of phytohaemagglutinin to lymphocytes is not dependent on the concurrent increases in intracellular cyclic A M P or prostaglandin El. However, the potent inhibition of this increase by adenosine and A M P is noteworthy. Although adenosine may inhibit uridine transport (Berlin & Oliver, 1979, it is difficult to account for the selective inhibition of the mitogen-induced increase in this way. An alternative possibility is that this is a very rapid indication of the known inhibitory effect of adenosine on the proliferation of lymphocytes unable to metabolize it by deamination (Hovi et a/.,1976). We thank the Wellcome Trust for financial support. Averner, M. J., Brock, M. L. & Jost, J.-P. (1972) J . Eiol. Chen?.247,413-417 Berlin, R. D. & Oliver, J. M. (1975) f n f . Rev. C.vfo/.42, 287-336 Hovi, T., Smyth, J. F.,Allison,A. C. &Williams, S. C. (1976) Clin. Exp. Immunol. 23,395-403 Kay, J. E.,Ahern,T.,Lindsay,V. J. &Sampson, J.(1975)Biocbim. Biopbys. Acta378,241-250 Ling, N. R. & Kay, J. E. (1975) Lyniphocyte Stburdation, 2ndedn., pp. 270-274,North Holland Publishing Co., Amsterdam Peters, J. H. & Hausen, P. (1971) Eur. 1. Biochem. 19,502-508 Smith,J. W.,Steiner,A. L.,Newberry, W. M. &Parker,C. W. (1971a)J. Clin. fnuest.50,432-441 Smith, J. W., Steiner, A. L. & Parker, C. W. (1971b)J. Clin. Inuesf. 50,442-448
Thymidine Uptake in F, Rat Cells Stimulated by Mitomycin-C-Treated Lymphocytes R A D H A SUBRAMANIAM and ALAN EBRINGER Department of Biochemistry, Queen Elizabeth College, London WS 7AH, U.K. When two populations of allogeneic lymphocytes are cultured together, a proliferative response occurs in a proportion of the cultured cells that can be estimated from the uptake of [3H]thymidine by the dividing cells, and this uptake is a measure of the immunological disparity between the two different populations of lymphocytes. It has been suggested that F, hybrid cells should not respond to inactivated parental cells because they share identical antigens; however, F, mice are capable of rejecting parental bone-marrow grafts (Cudkowicz & Bennett, 1971) and respond t o inactivated parental lymphocytes (Harrison & Paul, 1973). The present study was undertaken to assess whether FI responsiveness could be observed in rats, and to decrease the problem of genetic-strain disparity, F, litter animals were directly stimulated by parental lymphocytes. The rats used in the present study were adults of the strains Sprague-Dawley and Hooded Lister, and F1 hybrids derived from matings of these two strains were bred in the animal unit at this Institution. One-way-mixed lymphocyte cultures were established with spleen cells obtained from parental and individual F, littermates by using the technique of Peck &Bach (1973). The animals were exsanguinated and blood allowed to clot at room temperature, then kept at 4°C for 30min. The clotted blood was centrifuged for 1Smin at 2500rev./min and
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BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. [3H]T/zymidineuptake of parental and Fl hybrid rat spleen cells when stimulated with mitomycin-C-treatedparental cells The response of FI hybrid cells was compared with the response of the autologous parental male and female cells; Student’s t test was used and the statistical significance is indicated either at P
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stimulation bymitogen (Table 1). As little as 1OpM-adenosine inhibited thestimulation of uridine uptake by mitogen by 79% while decreasing the rate of uptake by unstimulated lymphocytes by only 24 %. Adenine or ATP at concentrations up to 1mM had much less effect on uridine uptake by lymphocytes incubated with or without mitogen. The addition of phytohaemagglutinin to lymphocytes also leads to a large but transient increase in intracellular prostaglandin Ez within the first hour, which can be prevented by prior addition of the prostaglandin synthesis inhibitors indomethacin or phenylbutazone (C. A. Phillips & J. E. Kay, unpublished work). However, Fig. 1 shows that concentrations of indomethacin or phenylbutazone that completely suppress the usual transient increase d o not affect the early increase in t3H]uridine uptake due to phytohaemagglutinin. These studies suggest that the rapid increase in [3H]uridine uptake after addition of phytohaemagglutinin to lymphocytes is not dependent on the concurrent increases in intracellular cyclic A M P or prostaglandin El. However, the potent inhibition of this increase by adenosine and A M P is noteworthy. Although adenosine may inhibit uridine transport (Berlin & Oliver, 1979, it is difficult to account for the selective inhibition of the mitogen-induced increase in this way. An alternative possibility is that this is a very rapid indication of the known inhibitory effect of adenosine on the proliferation of lymphocytes unable to metabolize it by deamination (Hovi et a/.,1976). We thank the Wellcome Trust for financial support. Averner, M. J., Brock, M. L. & Jost, J.-P. (1972) J . Eiol. Chen?.247,413-417 Berlin, R. D. & Oliver, J. M. (1975) f n f . Rev. C.vfo/.42, 287-336 Hovi, T., Smyth, J. F.,Allison,A. C. &Williams, S. C. (1976) Clin. Exp. Immunol. 23,395-403 Kay, J. E.,Ahern,T.,Lindsay,V. J. &Sampson, J.(1975)Biocbim. Biopbys. Acta378,241-250 Ling, N. R. & Kay, J. E. (1975) Lyniphocyte Stburdation, 2ndedn., pp. 270-274,North Holland Publishing Co., Amsterdam Peters, J. H. & Hausen, P. (1971) Eur. 1. Biochem. 19,502-508 Smith,J. W.,Steiner,A. L.,Newberry, W. M. &Parker,C. W. (1971a)J. Clin. fnuest.50,432-441 Smith, J. W., Steiner, A. L. & Parker, C. W. (1971b)J. Clin. Inuesf. 50,442-448
Thymidine Uptake in F, Rat Cells Stimulated by Mitomycin-C-Treated Lymphocytes R A D H A SUBRAMANIAM and ALAN EBRINGER Department of Biochemistry, Queen Elizabeth College, London WS 7AH, U.K. When two populations of allogeneic lymphocytes are cultured together, a proliferative response occurs in a proportion of the cultured cells that can be estimated from the uptake of [3H]thymidine by the dividing cells, and this uptake is a measure of the immunological disparity between the two different populations of lymphocytes. It has been suggested that F, hybrid cells should not respond to inactivated parental cells because they share identical antigens; however, F, mice are capable of rejecting parental bone-marrow grafts (Cudkowicz & Bennett, 1971) and respond t o inactivated parental lymphocytes (Harrison & Paul, 1973). The present study was undertaken to assess whether FI responsiveness could be observed in rats, and to decrease the problem of genetic-strain disparity, F, litter animals were directly stimulated by parental lymphocytes. The rats used in the present study were adults of the strains Sprague-Dawley and Hooded Lister, and F1 hybrids derived from matings of these two strains were bred in the animal unit at this Institution. One-way-mixed lymphocyte cultures were established with spleen cells obtained from parental and individual F, littermates by using the technique of Peck &Bach (1973). The animals were exsanguinated and blood allowed to clot at room temperature, then kept at 4°C for 30min. The clotted blood was centrifuged for 1Smin at 2500rev./min and
Vol. 6
1080
BIOCHEMICAL SOCIETY TRANSACTIONS
Table 1. [3H]T/zymidineuptake of parental and Fl hybrid rat spleen cells when stimulated with mitomycin-C-treatedparental cells The response of FI hybrid cells was compared with the response of the autologous parental male and female cells; Student’s t test was used and the statistical significance is indicated either at P
