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Differences in prostaglandin formation between thymocyte subpopulations
PROSTAGLANDINS
DIFFERENCES IN PROSTAGLANDIN FORMATION BETWEEN THYMOCYTE SUBPOPULATIONS Sara Bauminger Department of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel
ABSTRACT The concentration of prostaglandin E and the activity of prostaglandinsynthetase were determined in mature and immature mouse thymocytes. Hydrocortisone resistant thymocytes, or thymocytes separated from the immature cell population after agglutination of the latter by peanut agglutinin served as a source of mature thymocytes. It was found that mature thymocytes contain a much higher concentration of prostaglandin E and have an increased activity of prostaglandin synthetase than immature cells. INTRODUCTION Thymus lymphocytes are comprised of at least two distinct subpopulations, which differ in many features (1). The major group, amounting to about 85% of the total lymphocytes, is characterized by a high concentration of 8 antigen, the presence of TL antigen on its surface and sensitivity to treatment with hydrocortisone. The minor population, contains a low concentration of 8 antigen, lacks TL marker and is resistant to hydrocortisone (1). It is accepted that the minor population represents mature T lymphocytes, as it exhibits many of the functions of peripheral T lymphocytes, such as mitogen responsiveness and graft versus host activity (1,2,3). It has been reported that prostaglandins of the E type modify various immune responses, such as antibody release from antibody forming cells (4) and cytolytic activity of mouse lymphocytes (5). Since the latter activity is attributed to mature T lymphocytes (l), it was of interest to test whether significant differences exist in the level of prostaglandins of the E type in the two subpopulations, and if so, whether they are caused by activation of the PG-synthetase system. The results presented here show that mature T cells contain and synthesize a much higher concentration of prostaglandin E (PGE) than immature T cells.
SEPTEMBER 1978 VOL. 16 NO. 3
351
PROSTAGLANDINS
MATERIALS AND METHODS Antmale. Mice of the C57 B1/6J strain at the age of 8-10 weeks were obtained from the Animal Breeding Center of the Welzmann Institute. Preparation of cell homogenates. Thymocytes were prepared from untreated animals or from at&ala infected two days previously wlth hydrocortlaone acetate (125 mg/Kg body weight), The thymue glands were removed and single cell suspensions were prepared in a medium described previously (6). The separatlon of mature from immature thymocytes was performed according to Wlaner et al. (7). In this method, the immature thymocytes are agglutinated by peanut agglutlnin (PNA), and dlesoclated to single cells by addition of 0.3 M D-galactose; the mature cell6 are not agglutinated by PNA and can thus be separated from immature cells. Cell suspensions (20-46x106/ml) were homogentzed as described by Baumlnger, Zor and Llndner (6). Determination of PGE and PG synthetase. One portion of each homogenate was used for the determination of prostaglandln E (PGE). The other portlon, which served for the assay of PG synthetase, was incubated for 2 min. with arachldonlc acid (25 pg/ml) or wlthout exogenous substrate under the condltlons prevlouely described (8). All homogenates were extracted twke with 2 vol. ether: the ether layer was discarded and PGE concentration was determined in the aqueous phase l$v radlolmmunoassay (8) using an antiserum to PGE2 (6). Recovery of H-labeled proetaglandln E in the aqueous phase after thle extraction procedure was 78% + 3.3 ~E.M. (n=19). There was no difference in the extent The amount of PGE of recovery when different cell types were used. synthesized from exogenous arachldonlc acid was calculated by deducting PGE present in the no-substrate control from PGE formed in the presence of substrate. RESULTS AND DISCUSSION The concentration of prostaglandlns of E type and PG synthetase activity were determined In thymocytea obtalned from C57 B1/6J mice. The concentration of PGE was slgnlflcantly higher in hydrocortlsone resistant thymocytee (e.g. mature thymocytes) than in thymocytea of untreated anhnals (Table 1). It seems unllkeIy that the increased concentratton of PGE in hydrocortlsone rest&ant thymocytes is due to its direct effect on these lymphocytes, since hydrocortlsone has been shown to lnhlblt prostaglandln production (9). However, in order to exclude thls posslblllty, an addltlonal method, recently developed by Retsner et al. (7) was used for the separation of the two thymocyte subpopulatlons.
352
SEPTEMBER
1978 VOL. 16 NO. 3
PROSTAGLANDINS
This method is based on the difference in agglutinablllty of mature and immature thymocytes by PNA. PGE was determlPed In these two subpopulations of thymocytes and In unfractlonated thymocytes with or wlthout treatment wlth D galactose. TABLJI I Prostaglandln
concentration
and synthesis
ln mouse thymocytes
PGE synthesized
PGE concentration Treatment
Control Hydrocortlsons
@g/lo6 cells) 19.2 f 1.63 107
i6.7
Mean
l
SEM @g/lo’
cells/2
116.8 f
mln)
8.13
415.3 & 59.4
Prostaglandln concentration and prostaglandln syntI@slzed from exogenous arachldonlc acid were determined by radlolmmunoassay ln thymocytes obtained from control mice and from mice injected two days previously wlth hydrocortlsone acetate (125 mg/Kg body welght). Thymocytes from six mice were assayed ln each group in trlpllcate .
PGE concentration was slgnlflcantly higher ln the mature unagglutlnated thymocytes than ln lmmature or unfractlonated thymocytes, whether or not treated wlth D-galactose (Fig. 1). The elevated, levels of POE In mature thymocytee reeult from a rlae In PG-synthetase actlvlty (Table 1)‘and not from mere accumulation of prostaglandln ln the cells. R 1s not known however whether synthesla 1s enhanced In mature cells or sthnulatlon of exlstlng PG synthetase 1s lnvolved. The role played by endogenous prostaglandlns ln lymphocyte function has not been clarlfled, though It has been reported that exogenous PGE may lnhlblt cytolytlc actlvlty of mouse lymphocytes (5). It 1s posslble that a certain concentration of prostaglandlns 1s necessary for the actlvlty of T lymphocytes: whllst the amount present ln immature thymocytes may not be sufflclent to render them lmmunologlcally active, too high concentrations of prostaglandlns may be lnhlbltory. Whether PGE has any such physiological slgnlflcance remains to be determlned. Even so, PGE concentration may serve as a convenient marker for the recognltlon of mature thymocytes. SEPTEMBER 1978 VOL. 16 NO. 3
353
PROSTAGLAND’tiS
PIG. 1:. PGE concentration In mouse thymocyte subpopulatlons separated by peanut agglutlnln (PNA). I, untreated thymocytes; IL, thymocytes washed with 0.3 M D-galactose; III, thymocytes aggluttnated by PNA and dissociated by washing with 0.3 M D-galactose (immature thymocytes fraction); N, thymocytes not agglutinated by PNA, Mean values and S.E.M. (vertical brackets) are shown (n = 4). ACKNOWLEDGEMENTS I thank Mr. A. Almozntno and Mrs. S. Ovadia for technical assistance, Prof. N. Sharon and Mr. Y. Hsisner for a gift of PNA, the Upjohn Company for a gift of prostaglandln E3, and Prof. H. H. Llndnsr for helpful dlscusslons.
354
SEPTEMBER 1978 VOL. 16 NO. 3
PROSTAGLANDINS
HBFEHKNCES 1.
Greaves, M.F., Owen, J. J.T., and Haff, M. ‘IT and B lymphocytes: Origins, Properties and Holes in Immune Bespons~s~~, American Elsevier. pg. 27, 1973.
2.
Shortman, K., van Bohmer, H., Subpopulations of T-lymphocytes. 1975.
3.
Droege, W., and Zucker, Thymus. Transplantation
4.
Mebnon, K. L.. Bourne, H.H., Weinstein, Y., Shearer, G.M., S. Hemolytic Plaque Formation by Kram, J., and Baumlnger, Leukocytes -In Vitro. J. Cltn. Invest. 53:13, 1974.
5.
L.M. The Hole of Hetmey, C. S., Bourne, H. I?., and Lichtensteln, Cyclic 3’, 5’ Adenosine Monophosphate in the Specific Cytolyttc Activity of Lymphocytes. J. lmmunol. 108:1526, 1972.
6.
Bauminger, S., Zor, U., and Lindner, H. H. Radioimmunological Assay of Prostaglandin Synthetase Activity. Prostaglandins 4:313, 1973.
7.
Helsner, Y., Linker-Israeli, M., and Sharon, N. Separation of Mouse Thymocytes into Two Subpopulations by the Use of Peanut Agglutinin. Cellular Immunology 25:129, 1976.
8.
Baum inger, S., Lieberman, M. E., and Llndner, H. R SterotdIndependent Effect of Gonadotropin8 on Prostaglandin Syntheels In Bat G.raafian Follicles -In Vitro. Prostaglandins 9:753, 1975.
9.
Floman, N., and Zor, U. Mechanism of Steroid Actlon in Ocular Inflammation: tnhtbltlon of Prostaglandtn Production. Investigative Ophthalmology and Visual Science. 16:69, 1977.
SEPTEMBER
Llpp, J., and Hopper, K. Transplantation Hev. 25:163,
R Lymphocyte Subpopulations Rev. 25~3, 1975.
1978 VOL. 16 NO. 3
in the
DIFFERENCES IN PROSTAGLANDIN FORMATION BETWEEN THYMOCYTE SUBPOPULATIONS Sara Bauminger Department of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel
ABSTRACT The concentration of prostaglandin E and the activity of prostaglandinsynthetase were determined in mature and immature mouse thymocytes. Hydrocortisone resistant thymocytes, or thymocytes separated from the immature cell population after agglutination of the latter by peanut agglutinin served as a source of mature thymocytes. It was found that mature thymocytes contain a much higher concentration of prostaglandin E and have an increased activity of prostaglandin synthetase than immature cells. INTRODUCTION Thymus lymphocytes are comprised of at least two distinct subpopulations, which differ in many features (1). The major group, amounting to about 85% of the total lymphocytes, is characterized by a high concentration of 8 antigen, the presence of TL antigen on its surface and sensitivity to treatment with hydrocortisone. The minor population, contains a low concentration of 8 antigen, lacks TL marker and is resistant to hydrocortisone (1). It is accepted that the minor population represents mature T lymphocytes, as it exhibits many of the functions of peripheral T lymphocytes, such as mitogen responsiveness and graft versus host activity (1,2,3). It has been reported that prostaglandins of the E type modify various immune responses, such as antibody release from antibody forming cells (4) and cytolytic activity of mouse lymphocytes (5). Since the latter activity is attributed to mature T lymphocytes (l), it was of interest to test whether significant differences exist in the level of prostaglandins of the E type in the two subpopulations, and if so, whether they are caused by activation of the PG-synthetase system. The results presented here show that mature T cells contain and synthesize a much higher concentration of prostaglandin E (PGE) than immature T cells.
SEPTEMBER 1978 VOL. 16 NO. 3
351
PROSTAGLANDINS
MATERIALS AND METHODS Antmale. Mice of the C57 B1/6J strain at the age of 8-10 weeks were obtained from the Animal Breeding Center of the Welzmann Institute. Preparation of cell homogenates. Thymocytes were prepared from untreated animals or from at&ala infected two days previously wlth hydrocortlaone acetate (125 mg/Kg body weight), The thymue glands were removed and single cell suspensions were prepared in a medium described previously (6). The separatlon of mature from immature thymocytes was performed according to Wlaner et al. (7). In this method, the immature thymocytes are agglutinated by peanut agglutlnin (PNA), and dlesoclated to single cells by addition of 0.3 M D-galactose; the mature cell6 are not agglutinated by PNA and can thus be separated from immature cells. Cell suspensions (20-46x106/ml) were homogentzed as described by Baumlnger, Zor and Llndner (6). Determination of PGE and PG synthetase. One portion of each homogenate was used for the determination of prostaglandln E (PGE). The other portlon, which served for the assay of PG synthetase, was incubated for 2 min. with arachldonlc acid (25 pg/ml) or wlthout exogenous substrate under the condltlons prevlouely described (8). All homogenates were extracted twke with 2 vol. ether: the ether layer was discarded and PGE concentration was determined in the aqueous phase l$v radlolmmunoassay (8) using an antiserum to PGE2 (6). Recovery of H-labeled proetaglandln E in the aqueous phase after thle extraction procedure was 78% + 3.3 ~E.M. (n=19). There was no difference in the extent The amount of PGE of recovery when different cell types were used. synthesized from exogenous arachldonlc acid was calculated by deducting PGE present in the no-substrate control from PGE formed in the presence of substrate. RESULTS AND DISCUSSION The concentration of prostaglandlns of E type and PG synthetase activity were determined In thymocytea obtalned from C57 B1/6J mice. The concentration of PGE was slgnlflcantly higher in hydrocortlsone resistant thymocytee (e.g. mature thymocytes) than in thymocytea of untreated anhnals (Table 1). It seems unllkeIy that the increased concentratton of PGE in hydrocortlsone rest&ant thymocytes is due to its direct effect on these lymphocytes, since hydrocortlsone has been shown to lnhlblt prostaglandln production (9). However, in order to exclude thls posslblllty, an addltlonal method, recently developed by Retsner et al. (7) was used for the separation of the two thymocyte subpopulatlons.
352
SEPTEMBER
1978 VOL. 16 NO. 3
PROSTAGLANDINS
This method is based on the difference in agglutinablllty of mature and immature thymocytes by PNA. PGE was determlPed In these two subpopulations of thymocytes and In unfractlonated thymocytes with or wlthout treatment wlth D galactose. TABLJI I Prostaglandln
concentration
and synthesis
ln mouse thymocytes
PGE synthesized
PGE concentration Treatment
Control Hydrocortlsons
@g/lo6 cells) 19.2 f 1.63 107
i6.7
Mean
l
SEM @g/lo’
cells/2
116.8 f
mln)
8.13
415.3 & 59.4
Prostaglandln concentration and prostaglandln syntI@slzed from exogenous arachldonlc acid were determined by radlolmmunoassay ln thymocytes obtained from control mice and from mice injected two days previously wlth hydrocortlsone acetate (125 mg/Kg body welght). Thymocytes from six mice were assayed ln each group in trlpllcate .
PGE concentration was slgnlflcantly higher ln the mature unagglutlnated thymocytes than ln lmmature or unfractlonated thymocytes, whether or not treated wlth D-galactose (Fig. 1). The elevated, levels of POE In mature thymocytee reeult from a rlae In PG-synthetase actlvlty (Table 1)‘and not from mere accumulation of prostaglandln ln the cells. R 1s not known however whether synthesla 1s enhanced In mature cells or sthnulatlon of exlstlng PG synthetase 1s lnvolved. The role played by endogenous prostaglandlns ln lymphocyte function has not been clarlfled, though It has been reported that exogenous PGE may lnhlblt cytolytlc actlvlty of mouse lymphocytes (5). It 1s posslble that a certain concentration of prostaglandlns 1s necessary for the actlvlty of T lymphocytes: whllst the amount present ln immature thymocytes may not be sufflclent to render them lmmunologlcally active, too high concentrations of prostaglandlns may be lnhlbltory. Whether PGE has any such physiological slgnlflcance remains to be determlned. Even so, PGE concentration may serve as a convenient marker for the recognltlon of mature thymocytes. SEPTEMBER 1978 VOL. 16 NO. 3
353
PROSTAGLAND’tiS
PIG. 1:. PGE concentration In mouse thymocyte subpopulatlons separated by peanut agglutlnln (PNA). I, untreated thymocytes; IL, thymocytes washed with 0.3 M D-galactose; III, thymocytes aggluttnated by PNA and dissociated by washing with 0.3 M D-galactose (immature thymocytes fraction); N, thymocytes not agglutinated by PNA, Mean values and S.E.M. (vertical brackets) are shown (n = 4). ACKNOWLEDGEMENTS I thank Mr. A. Almozntno and Mrs. S. Ovadia for technical assistance, Prof. N. Sharon and Mr. Y. Hsisner for a gift of PNA, the Upjohn Company for a gift of prostaglandln E3, and Prof. H. H. Llndnsr for helpful dlscusslons.
354
SEPTEMBER 1978 VOL. 16 NO. 3
PROSTAGLANDINS
HBFEHKNCES 1.
Greaves, M.F., Owen, J. J.T., and Haff, M. ‘IT and B lymphocytes: Origins, Properties and Holes in Immune Bespons~s~~, American Elsevier. pg. 27, 1973.
2.
Shortman, K., van Bohmer, H., Subpopulations of T-lymphocytes. 1975.
3.
Droege, W., and Zucker, Thymus. Transplantation
4.
Mebnon, K. L.. Bourne, H.H., Weinstein, Y., Shearer, G.M., S. Hemolytic Plaque Formation by Kram, J., and Baumlnger, Leukocytes -In Vitro. J. Cltn. Invest. 53:13, 1974.
5.
L.M. The Hole of Hetmey, C. S., Bourne, H. I?., and Lichtensteln, Cyclic 3’, 5’ Adenosine Monophosphate in the Specific Cytolyttc Activity of Lymphocytes. J. lmmunol. 108:1526, 1972.
6.
Bauminger, S., Zor, U., and Lindner, H. H. Radioimmunological Assay of Prostaglandin Synthetase Activity. Prostaglandins 4:313, 1973.
7.
Helsner, Y., Linker-Israeli, M., and Sharon, N. Separation of Mouse Thymocytes into Two Subpopulations by the Use of Peanut Agglutinin. Cellular Immunology 25:129, 1976.
8.
Baum inger, S., Lieberman, M. E., and Llndner, H. R SterotdIndependent Effect of Gonadotropin8 on Prostaglandin Syntheels In Bat G.raafian Follicles -In Vitro. Prostaglandins 9:753, 1975.
9.
Floman, N., and Zor, U. Mechanism of Steroid Actlon in Ocular Inflammation: tnhtbltlon of Prostaglandtn Production. Investigative Ophthalmology and Visual Science. 16:69, 1977.
SEPTEMBER
Llpp, J., and Hopper, K. Transplantation Hev. 25:163,
R Lymphocyte Subpopulations Rev. 25~3, 1975.
1978 VOL. 16 NO. 3
in the