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Stimulation of phosphatidylinositol turnover by concanavalin a is not sufficient to activate mouse thymocytes
Vol. 141, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 657-664
December 15, 1986
STIMULATION OF PHOSPHATIDYLINOSITOL TURNOVER BY CONCANAVALIN A IS NOT SUFFICIENT TO ACTIVATE MOUSE THYMOCYTES Alan J. Feister and Harvey J. Sage Department of Biochemistry, Duke U n i v e r s i t y , and Veterans Administration Medical Center, Durham, NC 27705 Received August 29, 1986
Mouse thymocytes treated with the l e c t i n Concanavalin A do not p r o l i f e r a t e nor do they develop responsiveness to i n t e r l e u k i n 2. Co-treatment with Concanavalin A and e i t h e r l e c t i n - a c t i v a t e d splenocyte conditioned medium or phorbol ester caused increased i n t e r l e u k i n 2 receptor expression and proliferation. Under these conditions, l e c t i n alone stimulated a 3.4 fold increase in phosphatidylinositol turnover which was unaffected by the presence of conditioned medium. Phosphorylation of a 55 kD protein was stimulated in response to conditioned medium or phorbol ester, but not l e c t i n . These results indicate that stimulation of phosphoinositide turnover is not s u f f i c i e n t to activate thymocytes, and suggest that costimulating factors activate a kinase which is d i s t i n c t from protein kinase C, or a l t e r n a t i v e l y , activate protein kinase C through a process which is not coupled to phosphoinositide turnover. © 1986 Academic Press, Znc,
Activation
of
phosphoinositide
turnover
transmembrane s i g n a l l i n g mechanism for stimulated
breakdown of
second messengers: Ca2+ from the
has
a v a r i e t y of
polyphosphoinositides
reticulum,
hormones ( I ) .
gives
inositol-1,4,5-trisphosphate,
endoplasmic
b e e n proposed
rise
which
to
two
causes
and d i a c y l g l y c e r o l
as
a
Hormone potential
release
(DG), which
is
of an
a c t i v a t o r of protein kinase C (PKC). PKC has been proposed to be a mediator of activation reported
(2). to
In support of t h i s
activate
lectin
transient
phosphatidylinositol
association
of
T lymphocyte
hypothesis, mitogenic l e c t i n s (PI)
turnover
phosphatidylinositol-4,5-bisphosphate breakdown (6), stimulate
induced
PKC
with
(3,
4,
have been 5)
and have been found
the
cell
to
membrane (7).
ABBREVIATIONS 04, conditioned medium; Con A, Concanavalin A; d i a c y l g l y c e r o l ; IL2, i n t e r l e u k i n 2; IL2-R, i n t e r l e u k i n 2 receptor; p h o s p h a t i d y l i n o s i t o l ; PKC, protein kinase C.
657
and
DG, PI,
0006-291 X/86 $1.50 Copyright ~5 1986 by Academic Press, Inc. All rights of reproduction in any ,/orm reserved.
Vol. 141, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Furthermore, phorbol m%ristic acetate (PMA), which activates PKC by acting as a
DG analogue, is
ionophores (9).
strongly
comitogenic with
lectins
(8)
In some instances PMA is also mitogenic
and with
by i t s e l f
Ca2+
(10,11),
and can stimulate both the production of interleukin 2 (IL2) (12, 13) and the expression of IL2 receptors (IL2-R) (14). T lymphocyte mitogenesis occurs in two stages. stimulated
to increase
IL2-R expression
(priming).
First,
resting cells are
Primed cells
mitogenically stimulated following interaction of IL2 with its receptor.
are then
high a f f i n i t y
We have observed that priming of mouse thymocytes by Concanavalin A
(Con A) requires simultaneous exposure to Con A and coactivators present in conditioned medium from lectin-activated splenocytes cells,
Con A provides
only one of
two required
(15). signals
Since, for
in these
priming,
we
examined the effects of Con A and coactivators on PI turnover in order to determine whether Con A,
coactivators,
or
both together
were l i k e l y
to
activate PKC.
MATERIALS AND METHODS
Cells: Thymocytes were isolated from 3 to 7 week old Laboratories).
CBA/J mice (Jackson
Conditioned Medium r(CM): Lectin-depleted CM from 48 hour cultures of rat splenocytes (5 x I0 ° cells/ml) cultured with 5 ~g/ml Con A was employed as a source of growth factors. Proliferation Assay: Thymocytes were cultured at 5 x 105 cells/ml_ in 0.2 ml cultures in RPMI 1640 containing 10% fetal calf serum, 5 x I0 "b M 2-mercaptoethanol, and antibiotics. Four hours prior to harvesting, 0.5 pCi [3H] th~midine (20 Ci/mnol) was added to each culture and uptake of radiolabel was determined. IL2-R Expression: 2.5 x 106 cells were stained by sequential treatment with monoclonal rat anti-mouse IL2-R (7D4) (16) and fluoresceinated anti-rat k chain (17). Stained cells were analyzed on an Ortho Cytofluorographic Model 50H using linear amplifiers. Cells exhibiting fluorescence intensities above the region defined by unstimulated thymocytes treated with FITC-anti-rat k alone were considered to be 7D4+. Purified T cell blasts were used as a positive control. PI Turnover: Cells (5 x 105/ml) were preincubated for 2 hrs at 37°C~in phosphate-free minimal essentia~ medium (MEM). 500 ~Ci carrier-free ~Pi was added to aliquots of 3 x 10 cells, incubation was continued for 60 min, stimulants or controls were added, and incubation was continued for another 60 rain. Cells were centrifuged at 300 x g for 10 min at 5°C, washed with 2 ml ice-cold phosphate buffered saline and recentrifuged. Lipids were immediately extracted from the cell pellets (18), and phospholipids were
658
Vol. 141, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
separated by two dimensional thin layer chromatography (~9). spots were identified by comigration with standards and ~LPi was determined by liquid s c i n t i l l a t i o n counting.
Phospholipid incorporation
Protein Phosphorylation: Cells were suspended at 106/ml i n 6phosphate-free MEM and preincubated at 37°C for 60 m i n . Aliquots (10 cells) were labelled with 200 ~Ci of 32pi for 90 min at 3 7 ° C . Stimulants or controls were added and incubation continued for 10 min at 37°C. Cells were pelleted, dissociated in 50 ~l SDS sample buffer, and heated at 100°C for 5 m i n . Samples were analyzed by 7.5% PAGE SDS electrophoresis ( 2 0 ) and autoradiography. RESULTS At 106 cells/ml or less, thymocytes do not proliferate in response to Con A, CM, or PMA alone, but do respond to a combination of Con A and either CM or PMA (Table I)o
At a concentration
of
Con A which yields optimal
mitogenesis the maximum stimulation by Con A/PMA was t y p i c a l l y 50-80% of that seen with
Con A/CM.
PMA was an effective comitogen at
concentrations
of
0.5-10 ng/ml but was not mitogenic by i t s e l f at concentrations up to 100 ng/ml. We have previously observed (15) that thymocytes treated for
up to 48
hours with Con A alone do not proliferate when subsequently recultured IL2. and
Instead, CM, and
in
effective priming required simultaneous treatment with Con A proceeded concurrently
with
an
upregulation
of
IL2-R.
We
therefore compared the effects of PMA and CM on IL2-R expression (Table I I ) .
TABLE I.
Effect of Con A, CM, and PMA on thymocyte proliferation Stimulusa
cpmb
None
1,148 +
671
Con Ac
2,443 ~
316
CM c
1,715 +
360
PMAc
1,419 ~
582
Con A and CM
74,994 ~ 4,560
Con A and PMA
43,039 ~ 4,035
aThymocytes were incubated for 72 hours in the presence of additions as shown. bResults of triplicate determinations + standard deviation. CCon A = 3 ~g/ml; PMA = 10 ng/ml; CM=-3%. 659
Vol. 141, No. 2, 1986
TABLE I I .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect of Con A, CM, and PMA on IL2 receptor expression % 7D4+
Stimulants
Total b
High Density b
None
2.5
1.6
Con Ac
8.2
3.0
CMc
3.0
1.9
PMAc
3.5
0.6
Con A and CM
21.9
12.9
Con A and PMA
43.3
21.7
aThymocytes were cultured at 5 x 105 cells/ml for 48 hours in the presence of stimulants as shown prior to staining and cytofluorographic analysis. bTotal 7D4+ cells = channels 72-1000. High density region was a r b i t r a r i l y chosen to be from channels 151-1000. Purified T cells blasts used as a positive control were 99.5% 7D4+ with 90.6% high density. CCon A = 3 pg/ml; CM = 3%; PMA = I ng/ml.
Neither CM nor PMA s t i m u l a t e d IL2-R expression, and Con A alone caused only a small increase in the number o f c e l l s expressing IL2-R. PMA s t i m u l a t e d IL2-R expression in
the presence o f
less e f f e c t i v e than CM as a comitogen, i t
However, both CM and
Con A.
was more e f f e c t i v e
Although PMA was f o r coinduction
o f IL2-R.
TABLE I l l . Experiment
Effect of Con A and CM on 32p incorporation into PI Treatment
cpma
%
None Con Ab Con A and CMb
180 676 689
100 377 385
None Con A Con A and CM
156 401 354
100 258 227
None Con A Con A and CM
480 1,795 1,645
100 374 343
aResults are means of duplicate or t r i p l i c a t e determinations, corrected for r a d i o a c t i v i t y incorporated prior to addition of stimulants. Standard deviations were routinely less than 15%. bcon A = 3 ~g/ml; CM = 3%.
660
Vol. 141, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B
C
D
E
F
205
116 97.4
66
pp55 45 36 29 24
FIGURE I . Autoradiograph of thymocyte proteins labelled in response to Con A, CM and PMA. (A) no stimulant; (B) 3 pg/ml Con A; (C) 3% CM; (D) I0 ng/ml PMA; E) 3 ~g/ml Con A and I0 ng/ml PMA; (F) 3 ~g/ml Con A and 3% CM.
Previous studies metabolism
have
been
mitogenic ( 3 - 6 ) . but
not
of the e f f e c t s performed
Since,
sufficient
of mitogenic under
lectins
conditions
on p h o s p h o i n o s i t i d e
where
lectin
stimulus
stimulated
I n c o r p o r a t i o n of essentially
32p
for
effective
incorporation
radioactivity
identical
is
under the c o n d i t i o n s employed, Con A was a necessary IL2-R
upregulation
in
thymocytes,
the e f f e c t of Con A and CM on PI t u r n o v e r was examined (Table I I I ) . itself
alone
into
PI
into in
PI
the
by
2.5
to
presence of
Con A by 3.7
fold.
Con A and CM was
to t h a t seen in the presence o f Con A a l o n e ,
indicating
t h a t CM has no enhancing e f f e c t on PI t u r n o v e r . We next examined whether Con A, CM and PMA s t i m u l a t e d similar labelling
proteins.
C e l l s treated
of a 55 kD p r o t e i n
with either
(pp55)
(Figure 661
CM or I).
p h o s p h o r y l a t i o n of
PMA showed an increase Labelling
of
pp55 did
in not
Vol. 141, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
appear to be increased by treatment with Con A alone, nor did Con Anoticeably enhance the response to CM or PMA.
DISCUSSION We have examined the proposal that induction~ of involves PI stimulated
turnover-coupled PI
turnover
activation of
(presumably leading
PKC. to
IL2-R on T ljnnphocytes
We observed that activation
of
Con A
PKC) under
conditions where Con A was i n s u f f i c i e n t to prime thymocytes and caused only a small increase in IL2-R expression.
In contrast, simultaneous treatment with
Con A and the PKC activator PMA resulted in primed cells and large increases in IL2-R expression.
These results suggest that activation of PKC by Con A
induced PI turnover either does not occur, or is quantitatively insufficient to induce the large upregulation
of IL2 receptors required for priming.
It
has recently been reported that phytohemagglutinin stimulates transient (from 0 to 60 minutes) association of PKC with the membrane fraction in resting human peripheral
blood lymphocytes (7).
activation, membrane binding (21).
Mitogen-activated
of
PKC is
Although suggestive enhanced by elevated
lymphocytes have been reported
to
of
enzyme
Ca2+ levels have a
peak
i n t r a c e l l u l a r Ca2+ concentration of 250 nM (22), which is below the Ka for Ca2+ activation of PKC in the presence of DG in v i t r o (23), but is in the range
of
values at
which increased membrane binding
is
observed (21).
Although PKC may be transiently activated by lectins, our data indicate that this response i s , at least, insufficient to induce priming.
Activation of PKC
by PMA does co-induce IL2-R in the presence of l e c t i n , but, unlike l e c t i n , activation of PKC by PMA is probably prolonged (7).
We have also found that a
prolonged exposure of thymocytes to Con A (at least 10 hours) is required for co-activation with CM, suggesting that the i n t r a c e l l u l a r signals which are required are sustained over this period. We examined the p o s s i b i l i t y that cotreatment with Con A and ~ the activation of PI turnover beyond that seen with Con A alone.
enhanced Although
both CM and PMA act s i m i l a r l y in priming thymocytes in the presence of Con A 662
Vol, 141, No. 2, 1986 and stimulate
phosphorylation of
enhancing effect additional
of
activation
interleukin costimulators little
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
1 and with
pp55, we were unable to demonstrate any
CM on Con A induced PI of PKC through this IL2,
both of
turnover,
pathway.
which are
present
arguing against
We have observed that in
CM, can
Con A to upregulate IL2-R on thymocytes.
act
as
At present,
is known about the signalling mechanisms activated by these agents.
IL2 has also been suggested to act through PKC (24) but this point remains controversial
(25).
Our results are consistent
factors and PMA stimulate
parallel
with the notion that growth
pathways, resulting in the regulation of
the same key processes by two distinct
kinases.
Alternatively,
PKC may be
activated by growth factors through a process not involving PI turnover
(for
example, proteolytic conversion to the phospholipid independent form) (26, 27). ACKNOWLEDGMENTS- The authors wish to thank Dr. Ethan Shevach for the 7D4 antibody, and Dr. Allen Eckhardt for the anti-rat k chain. We also thank Ms. Curl ey Horton for technical assistance. T h i s work was supported by Veterans Administration General Medical Research Funds and by NIH Grant GM07184. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Berridge, M.J. (1984) Biochem. J., 22Q, 345-360. Ku, Y., Kishimoto, A., Takai, Y., Ogawa, Y;, Kimura, S., and Nishizuka, Y. (1981) J. Immunol., 127, 1375-1379. Fisher, D.B. and Mueller, G.C. (1968) Proc. Natl. Acad. Sci. USA, 60, 1396-1402. Fisher, D.B. and Mueller, G.C. (1971) Biochim. Biophys. Acta, 24__.88, 434-448. Maino, V.C., Hayman, M.J., and Crumpton, M.J. (1975) Biochem. J., 196, 247-252. Taylor, M.V., Metcalfe, J.C., Hesketh, T.R., Smith, G.A., and Moore, J.P. (1984) Nature (London), 312, 462-465. Farrar, W.L. and Ruscetti, F.W. (1986) J. Immunol., !36, 1266-1273. Mastro, A.M. and Mueller, G.C. (1974) Exp. Cell. Res., 88, 40-46. Truneh, A., Albert, F., Golstein, P., and Schmitt-Verhulst, A-M. (1985) Nature (London), 31.__33,318-320. Abb, J., Bayliss, G.J., and Deinhardt, F. (1979) J. Immunol., 122, 1639-1642. Orosz, C.F., Roopernian, D.C., and Bach, F.H. (1983) J. Immunol., 130, 1764-1769. Farrar, J.J., Mizel, S.B., Fuller-Farrar, J., Farrar, W.L., and Hilfiker, M.L. (1980) J. Immunol., 125, 793-798. Depper, J.M., Leonard, W.J., Kronke, M., Noguchi, P.D., Cunningham, R.E., Waldmann, T.R., and Greene, W.C. (1984) J. Immunol., 133, 3054-3061. Suzuki, T. and Cooper, M.D. (1985) J. Immunol., 134, ~TIF1-3119. Feister, A.J. and Sage, H.J., submitted for p u b l ~ t i o n . Malek, T.R., Robb, R.J., and Shevach, E.M. (1983) Proc. Natl. Acad. Sci. USA, 80, 5694-5698. Springer, T.A., Battacharya, A., Cardoza, J.T., and Sanchez-Madrid, F. (1982) Hybridoma, ~, 257-273. 663
Vol. 141, No. 2, 1986 18. 19. 20. 21. 22. 23. 24, 25. 26. 27.
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
B i l l a h , M.M. and Lapetina, E.G. (1982) d. Biol. Chem., 257, 12705-12708. Esko, J.D. and Raetz, C.R.H. (1980) J. Biol. Chem., 257~474-4480. Laemmli, U.K. (1970) Nature (London), 227, 680-685. Wolf, M., LeVine, H. I I I , May, W.W. d r . , Cuatrecasas, P., and Sayhoun, N. (1986) Nature (London), 317, 546-549. Tsien, R.Y., Pozzan, T., and Rink, T.J. (1982) Nature (London), 295, 68-7 I . Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U., and Nishizuka, Y. (1980) J. Biol. Chem., 255, 2273-2276. Far~ar, W.L. and Anderson, W.B. (1985) Nature (London), 315, 233-235. M i l l s , G.B., Stewart, D.J., Mellors, A., and Gelfand, E.W. (1986) J. Immunol., 136, 3019-3024. Kishimoto,'--A-., Kajikawa, N., Shiota, M., and Nishizuka, Y. (1983) J. Biol. Chem., 258, 1156-1164. Melloni, E., Pontremoli, S., Michetti, M., Sacco, 0., Sparatore, B., Salamino, F., and Horecker, B.L. (1985) Proc. Natl. Acad. Sci. USA, 82, 6435-6439.
664
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 657-664
December 15, 1986
STIMULATION OF PHOSPHATIDYLINOSITOL TURNOVER BY CONCANAVALIN A IS NOT SUFFICIENT TO ACTIVATE MOUSE THYMOCYTES Alan J. Feister and Harvey J. Sage Department of Biochemistry, Duke U n i v e r s i t y , and Veterans Administration Medical Center, Durham, NC 27705 Received August 29, 1986
Mouse thymocytes treated with the l e c t i n Concanavalin A do not p r o l i f e r a t e nor do they develop responsiveness to i n t e r l e u k i n 2. Co-treatment with Concanavalin A and e i t h e r l e c t i n - a c t i v a t e d splenocyte conditioned medium or phorbol ester caused increased i n t e r l e u k i n 2 receptor expression and proliferation. Under these conditions, l e c t i n alone stimulated a 3.4 fold increase in phosphatidylinositol turnover which was unaffected by the presence of conditioned medium. Phosphorylation of a 55 kD protein was stimulated in response to conditioned medium or phorbol ester, but not l e c t i n . These results indicate that stimulation of phosphoinositide turnover is not s u f f i c i e n t to activate thymocytes, and suggest that costimulating factors activate a kinase which is d i s t i n c t from protein kinase C, or a l t e r n a t i v e l y , activate protein kinase C through a process which is not coupled to phosphoinositide turnover. © 1986 Academic Press, Znc,
Activation
of
phosphoinositide
turnover
transmembrane s i g n a l l i n g mechanism for stimulated
breakdown of
second messengers: Ca2+ from the
has
a v a r i e t y of
polyphosphoinositides
reticulum,
hormones ( I ) .
gives
inositol-1,4,5-trisphosphate,
endoplasmic
b e e n proposed
rise
which
to
two
causes
and d i a c y l g l y c e r o l
as
a
Hormone potential
release
(DG), which
is
of an
a c t i v a t o r of protein kinase C (PKC). PKC has been proposed to be a mediator of activation reported
(2). to
In support of t h i s
activate
lectin
transient
phosphatidylinositol
association
of
T lymphocyte
hypothesis, mitogenic l e c t i n s (PI)
turnover
phosphatidylinositol-4,5-bisphosphate breakdown (6), stimulate
induced
PKC
with
(3,
4,
have been 5)
and have been found
the
cell
to
membrane (7).
ABBREVIATIONS 04, conditioned medium; Con A, Concanavalin A; d i a c y l g l y c e r o l ; IL2, i n t e r l e u k i n 2; IL2-R, i n t e r l e u k i n 2 receptor; p h o s p h a t i d y l i n o s i t o l ; PKC, protein kinase C.
657
and
DG, PI,
0006-291 X/86 $1.50 Copyright ~5 1986 by Academic Press, Inc. All rights of reproduction in any ,/orm reserved.
Vol. 141, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Furthermore, phorbol m%ristic acetate (PMA), which activates PKC by acting as a
DG analogue, is
ionophores (9).
strongly
comitogenic with
lectins
(8)
In some instances PMA is also mitogenic
and with
by i t s e l f
Ca2+
(10,11),
and can stimulate both the production of interleukin 2 (IL2) (12, 13) and the expression of IL2 receptors (IL2-R) (14). T lymphocyte mitogenesis occurs in two stages. stimulated
to increase
IL2-R expression
(priming).
First,
resting cells are
Primed cells
mitogenically stimulated following interaction of IL2 with its receptor.
are then
high a f f i n i t y
We have observed that priming of mouse thymocytes by Concanavalin A
(Con A) requires simultaneous exposure to Con A and coactivators present in conditioned medium from lectin-activated splenocytes cells,
Con A provides
only one of
two required
(15). signals
Since, for
in these
priming,
we
examined the effects of Con A and coactivators on PI turnover in order to determine whether Con A,
coactivators,
or
both together
were l i k e l y
to
activate PKC.
MATERIALS AND METHODS
Cells: Thymocytes were isolated from 3 to 7 week old Laboratories).
CBA/J mice (Jackson
Conditioned Medium r(CM): Lectin-depleted CM from 48 hour cultures of rat splenocytes (5 x I0 ° cells/ml) cultured with 5 ~g/ml Con A was employed as a source of growth factors. Proliferation Assay: Thymocytes were cultured at 5 x 105 cells/ml_ in 0.2 ml cultures in RPMI 1640 containing 10% fetal calf serum, 5 x I0 "b M 2-mercaptoethanol, and antibiotics. Four hours prior to harvesting, 0.5 pCi [3H] th~midine (20 Ci/mnol) was added to each culture and uptake of radiolabel was determined. IL2-R Expression: 2.5 x 106 cells were stained by sequential treatment with monoclonal rat anti-mouse IL2-R (7D4) (16) and fluoresceinated anti-rat k chain (17). Stained cells were analyzed on an Ortho Cytofluorographic Model 50H using linear amplifiers. Cells exhibiting fluorescence intensities above the region defined by unstimulated thymocytes treated with FITC-anti-rat k alone were considered to be 7D4+. Purified T cell blasts were used as a positive control. PI Turnover: Cells (5 x 105/ml) were preincubated for 2 hrs at 37°C~in phosphate-free minimal essentia~ medium (MEM). 500 ~Ci carrier-free ~Pi was added to aliquots of 3 x 10 cells, incubation was continued for 60 min, stimulants or controls were added, and incubation was continued for another 60 rain. Cells were centrifuged at 300 x g for 10 min at 5°C, washed with 2 ml ice-cold phosphate buffered saline and recentrifuged. Lipids were immediately extracted from the cell pellets (18), and phospholipids were
658
Vol. 141, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
separated by two dimensional thin layer chromatography (~9). spots were identified by comigration with standards and ~LPi was determined by liquid s c i n t i l l a t i o n counting.
Phospholipid incorporation
Protein Phosphorylation: Cells were suspended at 106/ml i n 6phosphate-free MEM and preincubated at 37°C for 60 m i n . Aliquots (10 cells) were labelled with 200 ~Ci of 32pi for 90 min at 3 7 ° C . Stimulants or controls were added and incubation continued for 10 min at 37°C. Cells were pelleted, dissociated in 50 ~l SDS sample buffer, and heated at 100°C for 5 m i n . Samples were analyzed by 7.5% PAGE SDS electrophoresis ( 2 0 ) and autoradiography. RESULTS At 106 cells/ml or less, thymocytes do not proliferate in response to Con A, CM, or PMA alone, but do respond to a combination of Con A and either CM or PMA (Table I)o
At a concentration
of
Con A which yields optimal
mitogenesis the maximum stimulation by Con A/PMA was t y p i c a l l y 50-80% of that seen with
Con A/CM.
PMA was an effective comitogen at
concentrations
of
0.5-10 ng/ml but was not mitogenic by i t s e l f at concentrations up to 100 ng/ml. We have previously observed (15) that thymocytes treated for
up to 48
hours with Con A alone do not proliferate when subsequently recultured IL2. and
Instead, CM, and
in
effective priming required simultaneous treatment with Con A proceeded concurrently
with
an
upregulation
of
IL2-R.
We
therefore compared the effects of PMA and CM on IL2-R expression (Table I I ) .
TABLE I.
Effect of Con A, CM, and PMA on thymocyte proliferation Stimulusa
cpmb
None
1,148 +
671
Con Ac
2,443 ~
316
CM c
1,715 +
360
PMAc
1,419 ~
582
Con A and CM
74,994 ~ 4,560
Con A and PMA
43,039 ~ 4,035
aThymocytes were incubated for 72 hours in the presence of additions as shown. bResults of triplicate determinations + standard deviation. CCon A = 3 ~g/ml; PMA = 10 ng/ml; CM=-3%. 659
Vol. 141, No. 2, 1986
TABLE I I .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect of Con A, CM, and PMA on IL2 receptor expression % 7D4+
Stimulants
Total b
High Density b
None
2.5
1.6
Con Ac
8.2
3.0
CMc
3.0
1.9
PMAc
3.5
0.6
Con A and CM
21.9
12.9
Con A and PMA
43.3
21.7
aThymocytes were cultured at 5 x 105 cells/ml for 48 hours in the presence of stimulants as shown prior to staining and cytofluorographic analysis. bTotal 7D4+ cells = channels 72-1000. High density region was a r b i t r a r i l y chosen to be from channels 151-1000. Purified T cells blasts used as a positive control were 99.5% 7D4+ with 90.6% high density. CCon A = 3 pg/ml; CM = 3%; PMA = I ng/ml.
Neither CM nor PMA s t i m u l a t e d IL2-R expression, and Con A alone caused only a small increase in the number o f c e l l s expressing IL2-R. PMA s t i m u l a t e d IL2-R expression in
the presence o f
less e f f e c t i v e than CM as a comitogen, i t
However, both CM and
Con A.
was more e f f e c t i v e
Although PMA was f o r coinduction
o f IL2-R.
TABLE I l l . Experiment
Effect of Con A and CM on 32p incorporation into PI Treatment
cpma
%
None Con Ab Con A and CMb
180 676 689
100 377 385
None Con A Con A and CM
156 401 354
100 258 227
None Con A Con A and CM
480 1,795 1,645
100 374 343
aResults are means of duplicate or t r i p l i c a t e determinations, corrected for r a d i o a c t i v i t y incorporated prior to addition of stimulants. Standard deviations were routinely less than 15%. bcon A = 3 ~g/ml; CM = 3%.
660
Vol. 141, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A
B
C
D
E
F
205
116 97.4
66
pp55 45 36 29 24
FIGURE I . Autoradiograph of thymocyte proteins labelled in response to Con A, CM and PMA. (A) no stimulant; (B) 3 pg/ml Con A; (C) 3% CM; (D) I0 ng/ml PMA; E) 3 ~g/ml Con A and I0 ng/ml PMA; (F) 3 ~g/ml Con A and 3% CM.
Previous studies metabolism
have
been
mitogenic ( 3 - 6 ) . but
not
of the e f f e c t s performed
Since,
sufficient
of mitogenic under
lectins
conditions
on p h o s p h o i n o s i t i d e
where
lectin
stimulus
stimulated
I n c o r p o r a t i o n of essentially
32p
for
effective
incorporation
radioactivity
identical
is
under the c o n d i t i o n s employed, Con A was a necessary IL2-R
upregulation
in
thymocytes,
the e f f e c t of Con A and CM on PI t u r n o v e r was examined (Table I I I ) . itself
alone
into
PI
into in
PI
the
by
2.5
to
presence of
Con A by 3.7
fold.
Con A and CM was
to t h a t seen in the presence o f Con A a l o n e ,
indicating
t h a t CM has no enhancing e f f e c t on PI t u r n o v e r . We next examined whether Con A, CM and PMA s t i m u l a t e d similar labelling
proteins.
C e l l s treated
of a 55 kD p r o t e i n
with either
(pp55)
(Figure 661
CM or I).
p h o s p h o r y l a t i o n of
PMA showed an increase Labelling
of
pp55 did
in not
Vol. 141, No. 2, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
appear to be increased by treatment with Con A alone, nor did Con Anoticeably enhance the response to CM or PMA.
DISCUSSION We have examined the proposal that induction~ of involves PI stimulated
turnover-coupled PI
turnover
activation of
(presumably leading
PKC. to
IL2-R on T ljnnphocytes
We observed that activation
of
Con A
PKC) under
conditions where Con A was i n s u f f i c i e n t to prime thymocytes and caused only a small increase in IL2-R expression.
In contrast, simultaneous treatment with
Con A and the PKC activator PMA resulted in primed cells and large increases in IL2-R expression.
These results suggest that activation of PKC by Con A
induced PI turnover either does not occur, or is quantitatively insufficient to induce the large upregulation
of IL2 receptors required for priming.
It
has recently been reported that phytohemagglutinin stimulates transient (from 0 to 60 minutes) association of PKC with the membrane fraction in resting human peripheral
blood lymphocytes (7).
activation, membrane binding (21).
Mitogen-activated
of
PKC is
Although suggestive enhanced by elevated
lymphocytes have been reported
to
of
enzyme
Ca2+ levels have a
peak
i n t r a c e l l u l a r Ca2+ concentration of 250 nM (22), which is below the Ka for Ca2+ activation of PKC in the presence of DG in v i t r o (23), but is in the range
of
values at
which increased membrane binding
is
observed (21).
Although PKC may be transiently activated by lectins, our data indicate that this response i s , at least, insufficient to induce priming.
Activation of PKC
by PMA does co-induce IL2-R in the presence of l e c t i n , but, unlike l e c t i n , activation of PKC by PMA is probably prolonged (7).
We have also found that a
prolonged exposure of thymocytes to Con A (at least 10 hours) is required for co-activation with CM, suggesting that the i n t r a c e l l u l a r signals which are required are sustained over this period. We examined the p o s s i b i l i t y that cotreatment with Con A and ~ the activation of PI turnover beyond that seen with Con A alone.
enhanced Although
both CM and PMA act s i m i l a r l y in priming thymocytes in the presence of Con A 662
Vol, 141, No. 2, 1986 and stimulate
phosphorylation of
enhancing effect additional
of
activation
interleukin costimulators little
BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS
1 and with
pp55, we were unable to demonstrate any
CM on Con A induced PI of PKC through this IL2,
both of
turnover,
pathway.
which are
present
arguing against
We have observed that in
CM, can
Con A to upregulate IL2-R on thymocytes.
act
as
At present,
is known about the signalling mechanisms activated by these agents.
IL2 has also been suggested to act through PKC (24) but this point remains controversial
(25).
Our results are consistent
factors and PMA stimulate
parallel
with the notion that growth
pathways, resulting in the regulation of
the same key processes by two distinct
kinases.
Alternatively,
PKC may be
activated by growth factors through a process not involving PI turnover
(for
example, proteolytic conversion to the phospholipid independent form) (26, 27). ACKNOWLEDGMENTS- The authors wish to thank Dr. Ethan Shevach for the 7D4 antibody, and Dr. Allen Eckhardt for the anti-rat k chain. We also thank Ms. Curl ey Horton for technical assistance. T h i s work was supported by Veterans Administration General Medical Research Funds and by NIH Grant GM07184. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Berridge, M.J. (1984) Biochem. J., 22Q, 345-360. Ku, Y., Kishimoto, A., Takai, Y., Ogawa, Y;, Kimura, S., and Nishizuka, Y. (1981) J. Immunol., 127, 1375-1379. Fisher, D.B. and Mueller, G.C. (1968) Proc. Natl. Acad. Sci. USA, 60, 1396-1402. Fisher, D.B. and Mueller, G.C. (1971) Biochim. Biophys. Acta, 24__.88, 434-448. Maino, V.C., Hayman, M.J., and Crumpton, M.J. (1975) Biochem. J., 196, 247-252. Taylor, M.V., Metcalfe, J.C., Hesketh, T.R., Smith, G.A., and Moore, J.P. (1984) Nature (London), 312, 462-465. Farrar, W.L. and Ruscetti, F.W. (1986) J. Immunol., !36, 1266-1273. Mastro, A.M. and Mueller, G.C. (1974) Exp. Cell. Res., 88, 40-46. Truneh, A., Albert, F., Golstein, P., and Schmitt-Verhulst, A-M. (1985) Nature (London), 31.__33,318-320. Abb, J., Bayliss, G.J., and Deinhardt, F. (1979) J. Immunol., 122, 1639-1642. Orosz, C.F., Roopernian, D.C., and Bach, F.H. (1983) J. Immunol., 130, 1764-1769. Farrar, J.J., Mizel, S.B., Fuller-Farrar, J., Farrar, W.L., and Hilfiker, M.L. (1980) J. Immunol., 125, 793-798. Depper, J.M., Leonard, W.J., Kronke, M., Noguchi, P.D., Cunningham, R.E., Waldmann, T.R., and Greene, W.C. (1984) J. Immunol., 133, 3054-3061. Suzuki, T. and Cooper, M.D. (1985) J. Immunol., 134, ~TIF1-3119. Feister, A.J. and Sage, H.J., submitted for p u b l ~ t i o n . Malek, T.R., Robb, R.J., and Shevach, E.M. (1983) Proc. Natl. Acad. Sci. USA, 80, 5694-5698. Springer, T.A., Battacharya, A., Cardoza, J.T., and Sanchez-Madrid, F. (1982) Hybridoma, ~, 257-273. 663
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B i l l a h , M.M. and Lapetina, E.G. (1982) d. Biol. Chem., 257, 12705-12708. Esko, J.D. and Raetz, C.R.H. (1980) J. Biol. Chem., 257~474-4480. Laemmli, U.K. (1970) Nature (London), 227, 680-685. Wolf, M., LeVine, H. I I I , May, W.W. d r . , Cuatrecasas, P., and Sayhoun, N. (1986) Nature (London), 317, 546-549. Tsien, R.Y., Pozzan, T., and Rink, T.J. (1982) Nature (London), 295, 68-7 I . Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U., and Nishizuka, Y. (1980) J. Biol. Chem., 255, 2273-2276. Far~ar, W.L. and Anderson, W.B. (1985) Nature (London), 315, 233-235. M i l l s , G.B., Stewart, D.J., Mellors, A., and Gelfand, E.W. (1986) J. Immunol., 136, 3019-3024. Kishimoto,'--A-., Kajikawa, N., Shiota, M., and Nishizuka, Y. (1983) J. Biol. Chem., 258, 1156-1164. Melloni, E., Pontremoli, S., Michetti, M., Sacco, 0., Sparatore, B., Salamino, F., and Horecker, B.L. (1985) Proc. Natl. Acad. Sci. USA, 82, 6435-6439.
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