- Email: [email protected]
Adenosine and adenine nucleotides are mitogenic for mouse thymocytes
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 1111-1116
August 14,1978
ADENCSINE ANDADENINENDCLEOTIDES ARE MITOGENICFORMOUSETHYMCCYTES Stephen Gregory and Milton Kern Institute
National
Received
of Arthritis, Metabolism, and Digestive Diseases National Institutes of Health Bethesda, Maryland 20014
June 22,1978
SUMMARY: Mouse thymocyte populations composedprincQally of e-bearing cells exhibited a fourfold or greated enhancement in DNA synthesis when cultured in the presence of adenosine or adenine nucleotides. In contrast Q-bearing cells derived from spleen nere markedly inhibited under the same circumstances, The effects of a variety of other nucleosides and nucleotides on DNA synthesis by spleen and thymus cells are also presented, Nucleosides and nucleotides have been implicated variety
of immuneprocesses.
in cyclic
AMP and/or cyclic
cyclic
GNPlevels
in response to mitogens such as
lymphocyte populations to cyclic
nucleotides, alterations
certain
(2) suggest
a relationship
nucleotide levels and immunefunction.
this conclusion (1,2).
of a
For example, studies demonstrating fluctuations
concanavalin A (1) or lipopolysaccharide intracellular
in the regulation
nucleotides added to culture
Besides effects
related
specifically
The response of fluids to
supports
cyclic
immunodeficiency diseases have been correlated
in adenosine metabolism which result
between
with
in aberrant intracellular
nucleotide levels and impared DNAsynthesis (3,4).
In experiments reported
here, a marked enhancement in DNA synthesis was observed in mousethymocyte cultures
incubated with adenosine or its phosphorylated derivatives.
sharp contrast,
In
DNAsynthesis by splenocytes or by splenic T cell-enriched
populations was inhibited
by these samecompounds. MATERIALSAND METHOE
Cell suspensions were prepared from the spleen and thymus of male 6-8 week old C3H/HeNmice (5) raised at the National Institute Cells were cultured
in medium199 supplemented nith IO$ fetal
of Health, calf serum,
0006-291x/78/0833-1111$01.00/0 1111
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
BIOCHEMICAL
Vol. 83, No. 3, 1978
35 $ml In all
penicillin, cases 4
x
200 &ml
lo6 cells/ml
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
glutamine and 20 ml+!Hepes buffer, culture
fluid
pli 7.5 (6,7).
were incubated 40 hr at 37'C in
the presence or absence of nucleosides and nucleotides.
The effects
number of compoundswere tested over a wide range of concentrations mM- 10 ti)
and a 1.0 mMfinal
throughout this study.
concentration
Each culture
of a (0.01
was judged optimal and used
received 5-10 pl L%$.hymidine for
the last 24 hours of the incubation period and the rate at DNA synthesis determined (7). T cell-enriched wool-adherent) of Trizio
(nylon
populations of mouse splenocytes were prepared by the method
and Cudkowicz (8).
method routinely O-positive
(nylon wool-nonadherent) and T cell-depleted
results
cells (g),
While fractionation
in a T cell-enriched
isolated T cell-enriched
of spleen cells by this
population comprised of 85-g@ populations were rechromato-
graphed on nylon wool columns in order to assure maximumenrichment. Nucleosides and nucleotides
used in this study were purchased from Sigma
Chemical Co., St. Louis, MO. ci+ 3 Thymidine (28.5 Ci/mMol) was obtained from New England Nuclear, Boston, Mass.; medium19 and fetal obtained from Grand Island Biologicals,
Grand Island,
calf
N.Y.
and mock mouseanti-B-serum were purchased from Litton
serum were
Mouse anti-e-serum
Bionetics,
Inc.,
Kensington, Md. REiULTSANDDISCUSSION Adenine derivatives
had a marked effect
on the rate of DNA synthesis
observed in spleen and thymus cell cultures. adenosine or .tienine
nucleotides
exhibited
Mouse thymocytes incubated with a four-
to fifteenfold
DNA synthesis whereas DNAsynthesis by splenocyte cultures manner was substantially nucleotides thymus cells.
failed
inhibited
to exhibit
(Table I).
treated
in the same
Other nucleosides and
the samedifferential
effect
on spleen and
For example, UTP and phosphorylated derivatives
stimulated DNAsynthesis by both spleen and thymus cultures, 5’4JMP and guanosine, on the other hand, had little
1112
increase in
of guanosine Uridine,
to no effect
on either
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS
Table I Mitogenic and anti-mitogenic nucleosides and nucleotides Additions
effects
of
on mouselymphocyte populations C3HS)rhymidineIncorporated Thymocyte Splenocyte
cm -Adenosine 5'-AMP
39,700 14,500
12,400 9,200
3’85’ CAMP dibutyryl 3’:5’
7,000 26,500
17,400 12,100
CAMP
ATP ^-
11,600
31,600 2,400
Uridine fj'-UMP
13,400 32,700 29,800
2,400 3,000
UTP
25,700 83,900 29,600
15,300 4,500 20,200
Guanosine GTP --
130,800
2,100
J-glycerophosphate 5’-GMP 3’:5’
wm
2,100
cGMP
Cytidine 5’-cMP
2,200
39,700 41,000
17,400
132,100
4,900 2,400
184,300 2,400
3,200
8,600
Cells (2 x 107) in 5 ml of mediumwere incubated 40 hr with or ithout 1.0 mMnucleoside or nucleotide. Each culture received 10 pCi [ Y-Hjthymidine for the last 24 hr of the Incubation period. The values shown are representative of those obtained in at least three and as many as eight different experiments, Duplicate samples within a given experiment agree to within { or - 1s.
cell population.
Cytidine
and 5’-CMF’ strongly
inhibited
the level
synthesis normally observed in splenocyte cultures while slightly DNA synthesis by thymocytes.
The effects
1113
of DNA enhancing
of nucleosides and nucleotides
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table II Adenine nucleotides
inhibit
by T cell-enriched
DNA synthesis
populations
of mousesplenocytes
IJ%
Additions
Original population
Thymidine Incorporated
T cell-enriched population
T cell-depleted population
cm
wm
34,400
3MO
72,200
‘j’-AME’
8,900
1,300
10,700
ATP
9,900
900
14,000
--
wm
T cell-enriched (nylon wool-nonadherent) and T cell-depleted (nylon wool-adherent) populations of mousesplenocytes were prepared by the method of Trizio and Cudkowicz (8). In order to enhance their separation, isolat populations were rechromotagraphed on nylon wool columns. 2 x 10f? cells in 0.5 ml mediumwere incubated 18 hr at 37% f or - 1.0 mM fj'-AMP or ATP. 5 yC1 (.?$thymidine was subsequently added and the cultures incubated an additional 24 hr.
were specific
insofar as other phosphorylated compoundssuch as)-glycero-
phosphate (see Table I), (data not shown) failed
glucosed-phosphate,
ribose and ribose-5-phosphate
to influence DNA synthesis by either spleen or
thymus cells, In order to define the cell type(s) the presence of adenine nucleotides, anti-B-serum plus complement prior Treatment with serum specific
stimulated to synthesize DNA in
thymocytes were treated to culturing
with mouse
with either 5'-AMP or ATP.
for O-antigen marklly
diminished the
elevated rate of DNA synthesis normally observed in thymocyte cultures incubated with adenine nucleotides.
On the other hand, treatment with mock
1114
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
mouseanti-B-serum plus complement had little response of thymocytes to 5'-AMP or ATP. e-positive
effect
Insofar
RESEARCH COMMUNICATIONS
on the subsequent
as most workers find that
cells comprise 95-$X3%of the mousethymocyte population (IO),
these results the majority
were not unexpected.
The results do indicate,
of cells stimulated by adenine derivatives
however, that
must have been
e-bearing lymphocytes rather than a minor , non-g-bearing population of cells present in thymus. Since spleen cell populations also contain O-bearing cells,
T cell-
enriched populations of splenocytes were prepared by nylon wool column filtration
(8) and tested for their
in Table II, inhibited
response to 5*-A!@ and ATP.
DNAsynthesis by the T cell-enriched
As shown
splenocyte fraction
was
by adenine nucleotides to the sameextent as was the synthesis
observed in either population.
the T cell-depleted
Filtration
fraction
or the original
splenic
through nylon wool columns, per se, had no effect
of thymocytes to respond to 5'-AMP or ATP and, therefore,
on the ability
cannot account for the failure
of adenine nucleotides to stimulate DNA
synthesis by splenic T cell-enriched
populations.
Inasmuch as T cell-
enriched spleen cell populations are comprised of at least 85-9@ g-positive cells
(9),
it is likely
that the population of e-bearing lymphocytes in
thymus which synthesizes DNAin response to incubation with adenine derivatives
is either absent or undetectable
These results
indicate
are capable of influencing lymphocyte populations.
in mousesplenic tissue,
that a number of nucleosides and nucleotides the level
of DNAsynthesis observed in mouse
It was of particular
interest
to note that while
DNA synthesis by B-bearing thymocytes was markedly enhanced in cultures incubated with adenine nucleotides, splenocytes was inhibited consistent cells
synthesis by cultures
by these samecompounds. Such findings
with the evidence of others (10) that indicate
residing
of e-bearing are
that e-positive
in the spleen are not the sameas those found in the thymus
and suggest, as well, an additional
meansfor T cell identification.
1115
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Another facet of this study which deserves special consideration derives from the fact that thymocyte cultures exhibit cells
the highest rate of DNA synthesis,
incubated with ATP routinely Since the response of thymus
to ATP was greater than the response to adenosine, it
conversion to adenosine was not a prerequisite mitogenic effect.
is likely
that
for ATP to exert its
Assuming that ATP remains in a phosphorylated form,
it is reasonable to suggest that DNAsynthesis is stimulated as a consequence of the interaction
between ATP and an external
site on the
plasma membraneof responding cells. ACKNOWLEDGEMENT Wewould like
to acknowledge the excellent
technical
assistance of
Mrs. B, Johnson, REFERENCES 1. ;:
Pastan, I. H., Johnson, C, S. and Anderson, W. B. (1975) Ann. Rev. Biochem. 44, 491-522. Watson, J. (1975) Transplant. Rev. 23, 221-249. Mills, G. C., Schmalstieg, F. C., Trimmer, K. B., Goldman, A. S. and Goldblum, R. M., (1976) Proc. Nat. Acad. Sci. U.S.A. 73,
2867-2871.
4. 5. 6. i: 9. 10.
Carson, D. A., Kaye, J. and Seegmiller, J. E. (1977) Proc. Nat. Acad. Sci. U.S.A. 74, 5677-5681. Swenson, R. M. and Kern, M. (1967) Proc. Nat. Acad. Sci. U.S.A.
57, 417-422.
Zimmerman,D. H, and Kern, M, (1973 J. Immi%oI. 111, 761-769. Zimmerman,D. H. and Kern, Ma (197 j J. Immunol. 111, 1326-1333. Trisio, D. and Cudkowicz, G. (1974 3 J. InmmoI. 113, 1093-1097. Julius, M, H., Simpson, E, and Herzenberg, L. A., (1973) Eur. J.
Immunol. 3, 645&49. Golub, E. S. (1977) The Cellular Sinauer Associates,
Inc.,
Basis of the Immune Response
Mass.
1116
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages 1111-1116
August 14,1978
ADENCSINE ANDADENINENDCLEOTIDES ARE MITOGENICFORMOUSETHYMCCYTES Stephen Gregory and Milton Kern Institute
National
Received
of Arthritis, Metabolism, and Digestive Diseases National Institutes of Health Bethesda, Maryland 20014
June 22,1978
SUMMARY: Mouse thymocyte populations composedprincQally of e-bearing cells exhibited a fourfold or greated enhancement in DNA synthesis when cultured in the presence of adenosine or adenine nucleotides. In contrast Q-bearing cells derived from spleen nere markedly inhibited under the same circumstances, The effects of a variety of other nucleosides and nucleotides on DNA synthesis by spleen and thymus cells are also presented, Nucleosides and nucleotides have been implicated variety
of immuneprocesses.
in cyclic
AMP and/or cyclic
cyclic
GNPlevels
in response to mitogens such as
lymphocyte populations to cyclic
nucleotides, alterations
certain
(2) suggest
a relationship
nucleotide levels and immunefunction.
this conclusion (1,2).
of a
For example, studies demonstrating fluctuations
concanavalin A (1) or lipopolysaccharide intracellular
in the regulation
nucleotides added to culture
Besides effects
related
specifically
The response of fluids to
supports
cyclic
immunodeficiency diseases have been correlated
in adenosine metabolism which result
between
with
in aberrant intracellular
nucleotide levels and impared DNAsynthesis (3,4).
In experiments reported
here, a marked enhancement in DNA synthesis was observed in mousethymocyte cultures
incubated with adenosine or its phosphorylated derivatives.
sharp contrast,
In
DNAsynthesis by splenocytes or by splenic T cell-enriched
populations was inhibited
by these samecompounds. MATERIALSAND METHOE
Cell suspensions were prepared from the spleen and thymus of male 6-8 week old C3H/HeNmice (5) raised at the National Institute Cells were cultured
in medium199 supplemented nith IO$ fetal
of Health, calf serum,
0006-291x/78/0833-1111$01.00/0 1111
Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
BIOCHEMICAL
Vol. 83, No. 3, 1978
35 $ml In all
penicillin, cases 4
x
200 &ml
lo6 cells/ml
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
glutamine and 20 ml+!Hepes buffer, culture
fluid
pli 7.5 (6,7).
were incubated 40 hr at 37'C in
the presence or absence of nucleosides and nucleotides.
The effects
number of compoundswere tested over a wide range of concentrations mM- 10 ti)
and a 1.0 mMfinal
throughout this study.
concentration
Each culture
of a (0.01
was judged optimal and used
received 5-10 pl L%$.hymidine for
the last 24 hours of the incubation period and the rate at DNA synthesis determined (7). T cell-enriched wool-adherent) of Trizio
(nylon
populations of mouse splenocytes were prepared by the method
and Cudkowicz (8).
method routinely O-positive
(nylon wool-nonadherent) and T cell-depleted
results
cells (g),
While fractionation
in a T cell-enriched
isolated T cell-enriched
of spleen cells by this
population comprised of 85-g@ populations were rechromato-
graphed on nylon wool columns in order to assure maximumenrichment. Nucleosides and nucleotides
used in this study were purchased from Sigma
Chemical Co., St. Louis, MO. ci+ 3 Thymidine (28.5 Ci/mMol) was obtained from New England Nuclear, Boston, Mass.; medium19 and fetal obtained from Grand Island Biologicals,
Grand Island,
calf
N.Y.
and mock mouseanti-B-serum were purchased from Litton
serum were
Mouse anti-e-serum
Bionetics,
Inc.,
Kensington, Md. REiULTSANDDISCUSSION Adenine derivatives
had a marked effect
on the rate of DNA synthesis
observed in spleen and thymus cell cultures. adenosine or .tienine
nucleotides
exhibited
Mouse thymocytes incubated with a four-
to fifteenfold
DNA synthesis whereas DNAsynthesis by splenocyte cultures manner was substantially nucleotides thymus cells.
failed
inhibited
to exhibit
(Table I).
treated
in the same
Other nucleosides and
the samedifferential
effect
on spleen and
For example, UTP and phosphorylated derivatives
stimulated DNAsynthesis by both spleen and thymus cultures, 5’4JMP and guanosine, on the other hand, had little
1112
increase in
of guanosine Uridine,
to no effect
on either
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS
Table I Mitogenic and anti-mitogenic nucleosides and nucleotides Additions
effects
of
on mouselymphocyte populations C3HS)rhymidineIncorporated Thymocyte Splenocyte
cm -Adenosine 5'-AMP
39,700 14,500
12,400 9,200
3’85’ CAMP dibutyryl 3’:5’
7,000 26,500
17,400 12,100
CAMP
ATP ^-
11,600
31,600 2,400
Uridine fj'-UMP
13,400 32,700 29,800
2,400 3,000
UTP
25,700 83,900 29,600
15,300 4,500 20,200
Guanosine GTP --
130,800
2,100
J-glycerophosphate 5’-GMP 3’:5’
wm
2,100
cGMP
Cytidine 5’-cMP
2,200
39,700 41,000
17,400
132,100
4,900 2,400
184,300 2,400
3,200
8,600
Cells (2 x 107) in 5 ml of mediumwere incubated 40 hr with or ithout 1.0 mMnucleoside or nucleotide. Each culture received 10 pCi [ Y-Hjthymidine for the last 24 hr of the Incubation period. The values shown are representative of those obtained in at least three and as many as eight different experiments, Duplicate samples within a given experiment agree to within { or - 1s.
cell population.
Cytidine
and 5’-CMF’ strongly
inhibited
the level
synthesis normally observed in splenocyte cultures while slightly DNA synthesis by thymocytes.
The effects
1113
of DNA enhancing
of nucleosides and nucleotides
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table II Adenine nucleotides
inhibit
by T cell-enriched
DNA synthesis
populations
of mousesplenocytes
IJ%
Additions
Original population
Thymidine Incorporated
T cell-enriched population
T cell-depleted population
cm
wm
34,400
3MO
72,200
‘j’-AME’
8,900
1,300
10,700
ATP
9,900
900
14,000
--
wm
T cell-enriched (nylon wool-nonadherent) and T cell-depleted (nylon wool-adherent) populations of mousesplenocytes were prepared by the method of Trizio and Cudkowicz (8). In order to enhance their separation, isolat populations were rechromotagraphed on nylon wool columns. 2 x 10f? cells in 0.5 ml mediumwere incubated 18 hr at 37% f or - 1.0 mM fj'-AMP or ATP. 5 yC1 (.?$thymidine was subsequently added and the cultures incubated an additional 24 hr.
were specific
insofar as other phosphorylated compoundssuch as)-glycero-
phosphate (see Table I), (data not shown) failed
glucosed-phosphate,
ribose and ribose-5-phosphate
to influence DNA synthesis by either spleen or
thymus cells, In order to define the cell type(s) the presence of adenine nucleotides, anti-B-serum plus complement prior Treatment with serum specific
stimulated to synthesize DNA in
thymocytes were treated to culturing
with mouse
with either 5'-AMP or ATP.
for O-antigen marklly
diminished the
elevated rate of DNA synthesis normally observed in thymocyte cultures incubated with adenine nucleotides.
On the other hand, treatment with mock
1114
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
mouseanti-B-serum plus complement had little response of thymocytes to 5'-AMP or ATP. e-positive
effect
Insofar
RESEARCH COMMUNICATIONS
on the subsequent
as most workers find that
cells comprise 95-$X3%of the mousethymocyte population (IO),
these results the majority
were not unexpected.
The results do indicate,
of cells stimulated by adenine derivatives
however, that
must have been
e-bearing lymphocytes rather than a minor , non-g-bearing population of cells present in thymus. Since spleen cell populations also contain O-bearing cells,
T cell-
enriched populations of splenocytes were prepared by nylon wool column filtration
(8) and tested for their
in Table II, inhibited
response to 5*-A!@ and ATP.
DNAsynthesis by the T cell-enriched
As shown
splenocyte fraction
was
by adenine nucleotides to the sameextent as was the synthesis
observed in either population.
the T cell-depleted
Filtration
fraction
or the original
splenic
through nylon wool columns, per se, had no effect
of thymocytes to respond to 5'-AMP or ATP and, therefore,
on the ability
cannot account for the failure
of adenine nucleotides to stimulate DNA
synthesis by splenic T cell-enriched
populations.
Inasmuch as T cell-
enriched spleen cell populations are comprised of at least 85-9@ g-positive cells
(9),
it is likely
that the population of e-bearing lymphocytes in
thymus which synthesizes DNAin response to incubation with adenine derivatives
is either absent or undetectable
These results
indicate
are capable of influencing lymphocyte populations.
in mousesplenic tissue,
that a number of nucleosides and nucleotides the level
of DNAsynthesis observed in mouse
It was of particular
interest
to note that while
DNA synthesis by B-bearing thymocytes was markedly enhanced in cultures incubated with adenine nucleotides, splenocytes was inhibited consistent cells
synthesis by cultures
by these samecompounds. Such findings
with the evidence of others (10) that indicate
residing
of e-bearing are
that e-positive
in the spleen are not the sameas those found in the thymus
and suggest, as well, an additional
meansfor T cell identification.
1115
BIOCHEMICAL
Vol. 83, No. 3, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Another facet of this study which deserves special consideration derives from the fact that thymocyte cultures exhibit cells
the highest rate of DNA synthesis,
incubated with ATP routinely Since the response of thymus
to ATP was greater than the response to adenosine, it
conversion to adenosine was not a prerequisite mitogenic effect.
is likely
that
for ATP to exert its
Assuming that ATP remains in a phosphorylated form,
it is reasonable to suggest that DNAsynthesis is stimulated as a consequence of the interaction
between ATP and an external
site on the
plasma membraneof responding cells. ACKNOWLEDGEMENT Wewould like
to acknowledge the excellent
technical
assistance of
Mrs. B, Johnson, REFERENCES 1. ;:
Pastan, I. H., Johnson, C, S. and Anderson, W. B. (1975) Ann. Rev. Biochem. 44, 491-522. Watson, J. (1975) Transplant. Rev. 23, 221-249. Mills, G. C., Schmalstieg, F. C., Trimmer, K. B., Goldman, A. S. and Goldblum, R. M., (1976) Proc. Nat. Acad. Sci. U.S.A. 73,
2867-2871.
4. 5. 6. i: 9. 10.
Carson, D. A., Kaye, J. and Seegmiller, J. E. (1977) Proc. Nat. Acad. Sci. U.S.A. 74, 5677-5681. Swenson, R. M. and Kern, M. (1967) Proc. Nat. Acad. Sci. U.S.A.
57, 417-422.
Zimmerman,D. H, and Kern, M, (1973 J. Immi%oI. 111, 761-769. Zimmerman,D. H. and Kern, Ma (197 j J. Immunol. 111, 1326-1333. Trisio, D. and Cudkowicz, G. (1974 3 J. InmmoI. 113, 1093-1097. Julius, M, H., Simpson, E, and Herzenberg, L. A., (1973) Eur. J.
Immunol. 3, 645&49. Golub, E. S. (1977) The Cellular Sinauer Associates,
Inc.,
Basis of the Immune Response
Mass.
1116