- Email: [email protected]
The elevation of mouse thymus cell cyclic adenosine monophosphate (cAMP) by lipopolysaccharide
The Elevation of Mouse Thymus Cell Cyclic Adenosine Monophosphate (cAMP) by Lipopolysaccharide Wen-Hui Tsien, Mayette Sampson, and Herbert Sheppard Abstract: The abxhty of certain hpopolysacchande (LPS) preparatzons to elevate cyclic adenosine monophosphate (cAMP) In mouse thymus cells ~n the presence of Ro 20-1724, 4-(3-butoxyl 4-methoxybenzyl)-2-~m~dazolididnone, was not related to the source of supply, bacterial strata, or method of extra.on Under the same condzt~ons adenosine ~sa potent ~mulator of thymus cell cAMP and is, of course, blocked by the further addition of theophylline When theophylline was added to the LPS preparations with Ro 20-1724, the cAMP produ~on was also blocked These studies suggested that the observed ~mulation of cAMP by LPS preparations was due to adenosine and/or its nucleot~des present as contaminants
Key Words: Mousethymocytes;cAMP, Lipopolysaccharide;Adenosine
INTRODUCTION Cyclic nucleotides have been reported to be involved in the proliferation and differentiation of lymphocytes induced by mitogens (Watson, 1976). Lipopolysaccharide preparations (of bacterial origin) at relatively high concentrations were shown to increase the cyclic adenosine monophosphate (cAMP) level of murine lymphocytes (Watson, 1976; Naylor et al., 1978). An effort to correlate the mitogentcity of several LPS preparations with their ability to elevate cAMP led to the finding that some m~togenic preparations failed to elevate cAMP. This prompted an investigation of a large number of LPS preparations that were derived from different bactenal species using either trichloroaceflc acid (TCA) or phenol extraction procedures and obtained from two commerical sources. It was found that only 8 of the 16 samples significantly elevated murine thymocyte cAMP levels at 25 pg/ml in the presence of a phosphodiesterase inhibitor (PDI), Ro 20-1724. This effect was blockable by theophylline, sugges~ng the presence of adenosine or a precusor in these active LPS preparations
Accepted February2, 1981 From the Departmentof Cell Biology, Roche ResearchCenter, Hoffmann-La Roche lnc, Nutley, New Jersey
Address requestsfor reprintsto Mrs Wen-Hw Tslen, Departmentof Cell Biology, Hoffmann-La Roche Inc, 340 Kingsland S~'eet, Nutley, NJ 07110 © ElsewerNorth Holland,lnc, 1981 52 VanderblltAve. New York, N Y lmmunopharmacology3, 253-258 (1981)
253 0162-310918110302530650250
254
W - H Tsien, M Sampson, and H Sheppard
MATERIALS AND METHODS
C57BL/6 male mice, 6 to 10 weeks old, were purchased from Jackson Lab, Bar Harbour, Maine LPS preparations were from Sigma and Difco Ro 2 0 - 1 7 2 4 was from Hoffmann La-Roche Inc. and theophylline was from Sigma. (2, 8) 3H-cyclic AMP of 3 0 - 5 0 Ci/mmol was from Amersham. Mouse thymus cell suspension was prepared as described previously (Tsien et al., 1980). The cells were suspended in a glucose-salt (GS) medium to a density of approximately 2 × 107 cells per milliliter. INCUBATION STUDIES
Aliquots of 0.25 ml of cell suspension were added to 0.25 ml of GS medium in 12 × 75 mm disposable glass culture tubes containing the PDI +- LPS. LPS preparations were dissolved in phosphate buffered saline (PBS) and the PDIs in GS. The samples were incubated at 37°C in a water shaker bath for 5 min. Reaction was stopped by heating in a boiling water bath for 3 rain Samples were stored frozen until ready for assay Cyclic AMP was measured by the competitive protein binding assay of Brown et al (1971) RESULTS
Mouse thymus cells incubated at 37°C for 5 min with LPS (Ehfco, L3922) in the presence of Ro 2 0 - 1 7 2 4 , a PDI, showed a dose-dependent rise of cAMP production (Fig. 1). No significant change was seen at lower concentrations. LPS preparations from Difco and Sigma, representing both TCA and phenol extraction, were similarly tested for such activity. It is apparent that the ability to elevate cAMP is not related to bacterial source, method of extraction, or commerical source but may be a nonpredictable event with particular lots (Table I). Since some LPS preparations were reported to contain substantial amounts of adenosine diphosphate (ADP) (Maclntyre et al., 1977), it was suspected that adenosine may be present as such or generated during incubation with the cells. Adenosine is a potent stimulator of mouse and guinea pig thymus cell cAMP production, which can be blocked by methylxanthines (Zenser, 1975, Fredholm et al., 1978). Peak stimulation by adenosine in the presence of 100 /zM Ro 2 0 - 1 7 2 4 was at 10 -5 M, and this was reduced to control level by the addition of 8 mM of theophylline (Fig. 2). This unique property of theophylline affords a way to distinguish a stimulatory activity of LPS from that of any containing adenosine. The stimulatory activity of the most active LPS preparation, Difco L3922, was measured in the presence of Ro 2 0 - 1 7 2 4 --theophylline. Stimulation was completely blocked by the addition of theophylline (Fig. 3) Other preparations that showed some elevation of cAMP were similarly blocked by the presence of theophylline (Fig. 4). A solution of L3922, I mg/ml, was dialyzed against PBS overnight. The dialyzed and the nondialyzed preparations were tested simultaneously for the cAMP elevating activity in the presence of Ro 2 0 - 1 7 2 4 and it was found that the dialyzed LPS had lost this activity and was inactive at 100 p~g/ml level. DISCUSSION The addition of mitogenic concentrations of LPS to spleen cells caused a transient rise of cGMP and, at higher concentrations, of cAMP (Watson, 1976) as well. Although a B-cell rnitogen, LPS Abbrevtations. LP$. lipopolysacchande;PDI phosphodiesteraseinhibitor; GS: glucose-salt,PBS" phosphate buffered saline,cAMP" cyclic adenosinemonophosphate; TCA" trichloroace~cacid; ADP adenosine diphosphate.
Lipolysaccharide on cAMP Elevation
255
/
IOC
-I
-I w b-
0
2
50-
o
./
E e,,
.,...@
//
ol
//,
1
I
I
OI
I0
I0
,
/,Lg L P S / m l
Figure 1 Thymus cells were incubated with increasing levels of LPS (L3922 from Difco) for 5 rain at 37°C in the presence of 0.1 mM Ro 20-1724 Results are the mean of quadruplet
samples was found to stimulate cAMP production more with thymus cells than spleen cells (Naylor et al., 1978) Our finding that not all of the LPS preparations possessed this ability to elevate thymus cell cAMP raises doubts about the involvement of cAMP in the mitogenic action of LPS Our doubts were augmented by an earlier report noting the presence of ADP in certain batches of LPS (Maclntyre et al., 1977} When different LPS preparations were tested for cAMP elevation with mouse thymocytes in the presence of Ro 2 0 - 1 7 2 4 , a PDI, the addition of theophylline completely blocked the elevation. Theophylline and isobutylmethylxanthine inhibited only the adenosine and not the prostaglandin E 1 mediated cAMP elevatmn with mouse thymocytes in the presence of Ro 2 0 - 1 7 2 4 or SQ65442 (Zenser, 1975) The stimulatmn of cAMP production, which can be blocked by methylxanthines, is a distinct characteristic of the adenosine response Such a response had been reported with several other cell types (Clark et al, 1974; Peck et al., 1974, Blume and Foster, 1975} and brain slices (Sattin and Rail, 1970, Schultz and Daly, 1973) While a potent stimulator of thymus cell cAMP, adenosine has only a weak effect on spleen cells, with only a twofold increase in cAMP at 1 mM (unpublished data) This could explain Naylor's finding that spleen cells are less sensitive to the cAMP elevation effect of LPS (Naylor et al, 1978) It is possible that the elevation of cAMP is through a non-purine component that causes release of adenosine or its denvatives from the thymus cells However, since the presence of ADP in LPS preparatmns has been reported, our results strongly suggest that the elevation of cAMP with murine lymphocytes is not an inherent property of LPS but that of a contaminant
Table 1
Stxmulatlon index a Extraction procedure
Source E coh
Manufacturer
Production
0111 0111 0111 0111
B4 B4 B4 B4
Sigma D1fco Sigma D1fco
0111 0128 0128 055 055 055 0127 0127
134 B12 B12 B5 B5 B5 B8 B8
Dffco Sigma Sigma Sigma Sigma Dffco Sigma Sigma
L-2630 L-3122 L-4130 L-3922 (Lot No 598159) L-3922 (666072) L-2755 L-4255 L-2880 L-4005 L-3923 L-3219 L-3880
Salmonella ententzdls
Sigma
L-3130
Salmonella ententldls
Sigma
L-4505
Salmonella typhosa
Sigma
L-3630
Salmonella typhosa
Sigma
L-5005
TCA b
Phenohc b
---
1 70** 0 97
1 40* 6 32*** 1 76*** 0 81"
--
0 83 1 14
--
1 36* --
2 12"**
1 45* 1 18
--
0 88 0 73** 1 32*** 1,23"*
a Different LPS preparatmns obtained from Sigma and Dlfco were tested for cAMP elevating actlwty at a concentratlon of 25/~g/ml The relative actlwty of each LPS was expressed m terms of stlmulalaon index, the ratm of cAMP produced in the presence of LPS over that of control containing PBS b *p < 0 05, **p < 0 01, ***p < 0 001
500
-
' ° ~ ~
400 ¢n _I ,.J t~J u
o
Ro 2 0 - 1 7 2 4
~o
300
u 2
200
tOO -- --I /-•. . . . . . 0 " "
~ 0~ -~ -8
TH EOPHYLLINE OR THEOPHYLLINE '.I- R 0 2 0 - 1 7 2 4
O ~ -7
--. -6
6 -5
i-4
M ADENOSINE
Figure 2 Thymocytes were incubated with adenosine, 10 -8 to 10 -4 M, i n the presence of 10 4 M Ro 2 0 - 1 7 2 4 or 8 x 10 -3 M theophyline or the combination for 5 rain at 37°C Results are the mean of quadruplet determination.
256
200
f°J
/
...1 .J w ¢.)
IIX u
/
w o E Q.
/
Ro20-1724
o
// THEOPHYLLINE + R020-1724
o~-
--// . . . . /I
~ I0
• /.Lq L 3 9 2 2
m
I I00
per ml
Figure 3 Thyrnocytes were incubated with LPS (L3922 from Difco) at concentrations of ] 0 - 2 0 0 i~g/rn! in the presence of 10-4 M Ro 2 0 - 1 7 2 4 or 8 × 10 -3 M theophylline + Ro 20 -1724 Results are the average of quadruplet samples.
o~
m ._1 I
I I x 10-4M R o 2 0 - 1 7 2 4
[~
200
8xlO-3M
Theophylhne
R o 2 0 - 1 7 2 4 + Theophyll,ne In ..J J w o
o o. =E I O 0 .J 0 o
N m ro
b')
Z
8
IL
. . . .
Figure 4 Thymocytes were incubated with different LPS preparations at 50 pg/rnl for 5 rnin at 37°C in the presence of10 - 4 M Ro 2 0 - 1 7 2 4 or 8 × 10- 3 M theophylline or both. Results are the mean of four samples. 257
258
W -H Tsien, M. Sampson, and H Sheppard
such as adenosine, or a precursor nudeohde such as ADP which is readily degraded into nucleoside by the thymus cells (Fredholm et al, 1978) REFERENCES
Blume AJ, Foster CA (1975) Mouse neuroblastoma adenylate cyclase Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity J Biol Chem 250.5003. Brown BL, Albano JDM, Ekins RP, Sghem AM (1971) A simple and sensitive saturation assay method for the measurement of adenosine 3',5'-cyclic monophosphate. Biochem J 121.561. Clark RB, Gross R, Su YF, Perkins, JP (1974) Regulation of adenosine 3',5'-monophosphate content in human astrocytma cells by adenosine and adenine nucleotide. J Biol Chem 249:5296. Fredholm BB, Sanberg G, Erstr6m U (1978) Cyclic AMP in freshly prepared thymocyte suspension Evidence for stimulation by endogenous adenosine. Biochem Pharmacol 27:2675. Maclntyre DS, Allen AP, Thome, KJI, Glauert AM, Gordon, JL (1977) Endotoxin-mduced platelet aggregation and secretion. J Cell Sci 28'211 Naylor PH, Camp C, Phillips AC, Thurman GB, Goldstein AL (1978) Effects of thymosin and lipopolysaccharide on murine lymphocyte cyclic AMP. J Immunol Meth 20:143. Peck WA, Carpenter J, Messinger K (1974) Cyclic 3',5'-adenosine monophosphate in isolated bone cells. II. Response to adenosine and parathyrold hormone Endocrinology 94:148. Sattin A, Rall TW (1970) The effect of adenosine and adenine nucleotides on the cyclic adenosine 3',-5'-phosphate content of guinea pig cerebral cortex slices Mol Pharmacol 6'13 Schultz J, Daly JD (1973) Cyclic adenosine 3',5'-monophosphate in guinea pig cerebral conical slices. J Biol Chem 248:853. Tsien W-H, Sampson M, Sheppard H (1980) Effects of ascorbic acid on 3H-thymldine incorporation by isolated mouse thymocytes, lmmunopharmacology 2' 117. Watson J (1976) The involvement of cyclic nucleotide metabolism in the initiation of lymphocyte proliferation induced by mitogens. J lmmunol 117:1656. Zenser TV (1975) Formation of adenosine 3',5'-monophosphate from adenosine in mouse thymocytes. Biochim Biophys Acta 404:202.
Key Words: Mousethymocytes;cAMP, Lipopolysaccharide;Adenosine
INTRODUCTION Cyclic nucleotides have been reported to be involved in the proliferation and differentiation of lymphocytes induced by mitogens (Watson, 1976). Lipopolysaccharide preparations (of bacterial origin) at relatively high concentrations were shown to increase the cyclic adenosine monophosphate (cAMP) level of murine lymphocytes (Watson, 1976; Naylor et al., 1978). An effort to correlate the mitogentcity of several LPS preparations with their ability to elevate cAMP led to the finding that some m~togenic preparations failed to elevate cAMP. This prompted an investigation of a large number of LPS preparations that were derived from different bactenal species using either trichloroaceflc acid (TCA) or phenol extraction procedures and obtained from two commerical sources. It was found that only 8 of the 16 samples significantly elevated murine thymocyte cAMP levels at 25 pg/ml in the presence of a phosphodiesterase inhibitor (PDI), Ro 20-1724. This effect was blockable by theophylline, sugges~ng the presence of adenosine or a precusor in these active LPS preparations
Accepted February2, 1981 From the Departmentof Cell Biology, Roche ResearchCenter, Hoffmann-La Roche lnc, Nutley, New Jersey
Address requestsfor reprintsto Mrs Wen-Hw Tslen, Departmentof Cell Biology, Hoffmann-La Roche Inc, 340 Kingsland S~'eet, Nutley, NJ 07110 © ElsewerNorth Holland,lnc, 1981 52 VanderblltAve. New York, N Y lmmunopharmacology3, 253-258 (1981)
253 0162-310918110302530650250
254
W - H Tsien, M Sampson, and H Sheppard
MATERIALS AND METHODS
C57BL/6 male mice, 6 to 10 weeks old, were purchased from Jackson Lab, Bar Harbour, Maine LPS preparations were from Sigma and Difco Ro 2 0 - 1 7 2 4 was from Hoffmann La-Roche Inc. and theophylline was from Sigma. (2, 8) 3H-cyclic AMP of 3 0 - 5 0 Ci/mmol was from Amersham. Mouse thymus cell suspension was prepared as described previously (Tsien et al., 1980). The cells were suspended in a glucose-salt (GS) medium to a density of approximately 2 × 107 cells per milliliter. INCUBATION STUDIES
Aliquots of 0.25 ml of cell suspension were added to 0.25 ml of GS medium in 12 × 75 mm disposable glass culture tubes containing the PDI +- LPS. LPS preparations were dissolved in phosphate buffered saline (PBS) and the PDIs in GS. The samples were incubated at 37°C in a water shaker bath for 5 min. Reaction was stopped by heating in a boiling water bath for 3 rain Samples were stored frozen until ready for assay Cyclic AMP was measured by the competitive protein binding assay of Brown et al (1971) RESULTS
Mouse thymus cells incubated at 37°C for 5 min with LPS (Ehfco, L3922) in the presence of Ro 2 0 - 1 7 2 4 , a PDI, showed a dose-dependent rise of cAMP production (Fig. 1). No significant change was seen at lower concentrations. LPS preparations from Difco and Sigma, representing both TCA and phenol extraction, were similarly tested for such activity. It is apparent that the ability to elevate cAMP is not related to bacterial source, method of extraction, or commerical source but may be a nonpredictable event with particular lots (Table I). Since some LPS preparations were reported to contain substantial amounts of adenosine diphosphate (ADP) (Maclntyre et al., 1977), it was suspected that adenosine may be present as such or generated during incubation with the cells. Adenosine is a potent stimulator of mouse and guinea pig thymus cell cAMP production, which can be blocked by methylxanthines (Zenser, 1975, Fredholm et al., 1978). Peak stimulation by adenosine in the presence of 100 /zM Ro 2 0 - 1 7 2 4 was at 10 -5 M, and this was reduced to control level by the addition of 8 mM of theophylline (Fig. 2). This unique property of theophylline affords a way to distinguish a stimulatory activity of LPS from that of any containing adenosine. The stimulatory activity of the most active LPS preparation, Difco L3922, was measured in the presence of Ro 2 0 - 1 7 2 4 --theophylline. Stimulation was completely blocked by the addition of theophylline (Fig. 3) Other preparations that showed some elevation of cAMP were similarly blocked by the presence of theophylline (Fig. 4). A solution of L3922, I mg/ml, was dialyzed against PBS overnight. The dialyzed and the nondialyzed preparations were tested simultaneously for the cAMP elevating activity in the presence of Ro 2 0 - 1 7 2 4 and it was found that the dialyzed LPS had lost this activity and was inactive at 100 p~g/ml level. DISCUSSION The addition of mitogenic concentrations of LPS to spleen cells caused a transient rise of cGMP and, at higher concentrations, of cAMP (Watson, 1976) as well. Although a B-cell rnitogen, LPS Abbrevtations. LP$. lipopolysacchande;PDI phosphodiesteraseinhibitor; GS: glucose-salt,PBS" phosphate buffered saline,cAMP" cyclic adenosinemonophosphate; TCA" trichloroace~cacid; ADP adenosine diphosphate.
Lipolysaccharide on cAMP Elevation
255
/
IOC
-I
-I w b-
0
2
50-
o
./
E e,,
.,...@
//
ol
//,
1
I
I
OI
I0
I0
,
/,Lg L P S / m l
Figure 1 Thymus cells were incubated with increasing levels of LPS (L3922 from Difco) for 5 rain at 37°C in the presence of 0.1 mM Ro 20-1724 Results are the mean of quadruplet
samples was found to stimulate cAMP production more with thymus cells than spleen cells (Naylor et al., 1978) Our finding that not all of the LPS preparations possessed this ability to elevate thymus cell cAMP raises doubts about the involvement of cAMP in the mitogenic action of LPS Our doubts were augmented by an earlier report noting the presence of ADP in certain batches of LPS (Maclntyre et al., 1977} When different LPS preparations were tested for cAMP elevation with mouse thymocytes in the presence of Ro 2 0 - 1 7 2 4 , a PDI, the addition of theophylline completely blocked the elevation. Theophylline and isobutylmethylxanthine inhibited only the adenosine and not the prostaglandin E 1 mediated cAMP elevatmn with mouse thymocytes in the presence of Ro 2 0 - 1 7 2 4 or SQ65442 (Zenser, 1975) The stimulatmn of cAMP production, which can be blocked by methylxanthines, is a distinct characteristic of the adenosine response Such a response had been reported with several other cell types (Clark et al, 1974; Peck et al., 1974, Blume and Foster, 1975} and brain slices (Sattin and Rail, 1970, Schultz and Daly, 1973) While a potent stimulator of thymus cell cAMP, adenosine has only a weak effect on spleen cells, with only a twofold increase in cAMP at 1 mM (unpublished data) This could explain Naylor's finding that spleen cells are less sensitive to the cAMP elevation effect of LPS (Naylor et al, 1978) It is possible that the elevation of cAMP is through a non-purine component that causes release of adenosine or its denvatives from the thymus cells However, since the presence of ADP in LPS preparatmns has been reported, our results strongly suggest that the elevation of cAMP with murine lymphocytes is not an inherent property of LPS but that of a contaminant
Table 1
Stxmulatlon index a Extraction procedure
Source E coh
Manufacturer
Production
0111 0111 0111 0111
B4 B4 B4 B4
Sigma D1fco Sigma D1fco
0111 0128 0128 055 055 055 0127 0127
134 B12 B12 B5 B5 B5 B8 B8
Dffco Sigma Sigma Sigma Sigma Dffco Sigma Sigma
L-2630 L-3122 L-4130 L-3922 (Lot No 598159) L-3922 (666072) L-2755 L-4255 L-2880 L-4005 L-3923 L-3219 L-3880
Salmonella ententzdls
Sigma
L-3130
Salmonella ententldls
Sigma
L-4505
Salmonella typhosa
Sigma
L-3630
Salmonella typhosa
Sigma
L-5005
TCA b
Phenohc b
---
1 70** 0 97
1 40* 6 32*** 1 76*** 0 81"
--
0 83 1 14
--
1 36* --
2 12"**
1 45* 1 18
--
0 88 0 73** 1 32*** 1,23"*
a Different LPS preparatmns obtained from Sigma and Dlfco were tested for cAMP elevating actlwty at a concentratlon of 25/~g/ml The relative actlwty of each LPS was expressed m terms of stlmulalaon index, the ratm of cAMP produced in the presence of LPS over that of control containing PBS b *p < 0 05, **p < 0 01, ***p < 0 001
500
-
' ° ~ ~
400 ¢n _I ,.J t~J u
o
Ro 2 0 - 1 7 2 4
~o
300
u 2
200
tOO -- --I /-•. . . . . . 0 " "
~ 0~ -~ -8
TH EOPHYLLINE OR THEOPHYLLINE '.I- R 0 2 0 - 1 7 2 4
O ~ -7
--. -6
6 -5
i-4
M ADENOSINE
Figure 2 Thymocytes were incubated with adenosine, 10 -8 to 10 -4 M, i n the presence of 10 4 M Ro 2 0 - 1 7 2 4 or 8 x 10 -3 M theophyline or the combination for 5 rain at 37°C Results are the mean of quadruplet determination.
256
200
f°J
/
...1 .J w ¢.)
IIX u
/
w o E Q.
/
Ro20-1724
o
// THEOPHYLLINE + R020-1724
o~-
--// . . . . /I
~ I0
• /.Lq L 3 9 2 2
m
I I00
per ml
Figure 3 Thyrnocytes were incubated with LPS (L3922 from Difco) at concentrations of ] 0 - 2 0 0 i~g/rn! in the presence of 10-4 M Ro 2 0 - 1 7 2 4 or 8 × 10 -3 M theophylline + Ro 20 -1724 Results are the average of quadruplet samples.
o~
m ._1 I
I I x 10-4M R o 2 0 - 1 7 2 4
[~
200
8xlO-3M
Theophylhne
R o 2 0 - 1 7 2 4 + Theophyll,ne In ..J J w o
o o. =E I O 0 .J 0 o
N m ro
b')
Z
8
IL
. . . .
Figure 4 Thymocytes were incubated with different LPS preparations at 50 pg/rnl for 5 rnin at 37°C in the presence of10 - 4 M Ro 2 0 - 1 7 2 4 or 8 × 10- 3 M theophylline or both. Results are the mean of four samples. 257
258
W -H Tsien, M. Sampson, and H Sheppard
such as adenosine, or a precursor nudeohde such as ADP which is readily degraded into nucleoside by the thymus cells (Fredholm et al, 1978) REFERENCES
Blume AJ, Foster CA (1975) Mouse neuroblastoma adenylate cyclase Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity J Biol Chem 250.5003. Brown BL, Albano JDM, Ekins RP, Sghem AM (1971) A simple and sensitive saturation assay method for the measurement of adenosine 3',5'-cyclic monophosphate. Biochem J 121.561. Clark RB, Gross R, Su YF, Perkins, JP (1974) Regulation of adenosine 3',5'-monophosphate content in human astrocytma cells by adenosine and adenine nucleotide. J Biol Chem 249:5296. Fredholm BB, Sanberg G, Erstr6m U (1978) Cyclic AMP in freshly prepared thymocyte suspension Evidence for stimulation by endogenous adenosine. Biochem Pharmacol 27:2675. Maclntyre DS, Allen AP, Thome, KJI, Glauert AM, Gordon, JL (1977) Endotoxin-mduced platelet aggregation and secretion. J Cell Sci 28'211 Naylor PH, Camp C, Phillips AC, Thurman GB, Goldstein AL (1978) Effects of thymosin and lipopolysaccharide on murine lymphocyte cyclic AMP. J Immunol Meth 20:143. Peck WA, Carpenter J, Messinger K (1974) Cyclic 3',5'-adenosine monophosphate in isolated bone cells. II. Response to adenosine and parathyrold hormone Endocrinology 94:148. Sattin A, Rall TW (1970) The effect of adenosine and adenine nucleotides on the cyclic adenosine 3',-5'-phosphate content of guinea pig cerebral cortex slices Mol Pharmacol 6'13 Schultz J, Daly JD (1973) Cyclic adenosine 3',5'-monophosphate in guinea pig cerebral conical slices. J Biol Chem 248:853. Tsien W-H, Sampson M, Sheppard H (1980) Effects of ascorbic acid on 3H-thymldine incorporation by isolated mouse thymocytes, lmmunopharmacology 2' 117. Watson J (1976) The involvement of cyclic nucleotide metabolism in the initiation of lymphocyte proliferation induced by mitogens. J lmmunol 117:1656. Zenser TV (1975) Formation of adenosine 3',5'-monophosphate from adenosine in mouse thymocytes. Biochim Biophys Acta 404:202.