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Cyclic adenosine 3′,5′-monophosphate response to dopamine in mouse lymphoid cells and cell lines
Life Sciences, Vol. 40, pp. 2385-2392 Printed in the U.S.A.
Pergamon Journals
CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE RESPONSE TO DOPAMINE IN MOUSE LYMPHOID CELLS AND CELL LINES E. Kouassi, Y.S. Li, M. Flacher and J.P. Revillard Laboratoire d'immunologie, INSERM U.80, CNRS UA 1177, UCBL, HSpital E. Herriot 690.37 Lyon Cedex 03, France (Received
in final form April 2, 1987)
Summary Cyclic adenosine 3',5'-monophosphate responses to dopamine and isoproterenol were studied in mouse and rat spleen, thymus, lymph nodes and Peyer's patches lymphocytes and in 7 mouse cell lines of T- and B-lymphoid derivation. The responses of normal cells to dopamine were moderate, of the same extent, but selective to spleen and thymus in mouse, and to spleen and lymph nodes in rat. The YAC-1 T Iymphorna cell line was sensitive to dopamine with a higher magnitude than normal lymphoid cells. Dopamine was less potent than isoproterenol in all celts, and whereas dopaminesensitive and isoproterenol-sensitive celts, or dopamine-insensitive and isoproterenol-insensitive cells were found, no cell type was dopamine-sensitive and isoproterenol-insensitive. Altogether, the.,~e results suggest that only a small subset of lymphocytes is susceptible to the cAMP-elevating action of dopamine. Lymphocyte responses may be regulated by catecho|amines released by nerve fibers which end adjacent to immune cells in thymus-dependent areas of lymphoid tissues (1). Many of the immunological effects of the 6-adrenergic catecholamines as well as those of numerous hormones, neurotransmitters and drugs, result from activation of the enzyme adenytate cyclase and the subsequently increased formation of intracellutar cyclic adenosine 3',5'-monophosphate (cAMP) which acts then as a "second messenger" (2-4). Dopamine (DA) can act on a-and 6-adrenergic receptors (5) and on two main types of specific receptors classified in respect to their association with adenylate cyclase activity (6): D-1 receptors which can cause an increase in adenylate cyclase activity, and 0-2 receptors which are not stimulatory [inked to the adenylate cyclase. Stimulation of D-2 receptors can in some cases even decrease adenylate cyclase activity (7). The existence of D-2 DA receptors have been postulated on mammalian lymphocytes (8) with B-cell selectivity (9) by using the binding of 3H-spiroperidol as a specific ligand. However, and although the number of binding sites in lymphocytes was affected in certain pathological conditions (10-16), a number of studies (15, 17-22) failed to confirm that 3H-spiroperido! binds to a specific DA receptor comparable to that of the brain (23) or peripheral (24) DA receptors. These studies suggested that the labelled ligand may be taken up by lysosomes. Conversely, Stepien et al. (25) showed that addition of DA at micromolar concentrations increased cAMP concentrations in the rat spleen cells, suggesting the presence of D-1 DA receptors on these cells although Shaskan et al. (22) could not demonstrate displaceable binding of a relatively selective D-1 DA receptor agonist 3H-2-amino-6,7-dihydroxy-l,2,%4-tetrahydronaphthalene (ADTN) on mouse thymus or spleen cells. 002/4-3025/87 $3.00 + .00 Copyright
(c) 1987 Pergamon Journals
Ltd.
2386
Lymphocyte cAMP Response to DA
Vol. 40, No. 25, 1987
In view of the important immunoregulatory role of cAMP in immunocytes, and the remarkable differences in the cAMP response to various hormones in subpopulations of ]ymphocytes related to their cell type and state of maturation and differentiation (2630), we investigated the cAMP response to DA in mouse Iymphocytes from spleen, thymus and Peyer's patches, and in various mouse T-and B-lymphoma cell lines. For comparative purposes, responses of rat spleen, thymus, lymph nodes and Peyer's patches cells~ and responses to the ~-adrenergie agonist isoproterenol (ISO) were studied. Materials and Methods Animals. Male and female 6-12 weeks old BALB/c or C57B1/63 mice and male 6-8 weeks old Sprague-Dawley rats served as normal cell donors. BALB/c mice were bred in our laboratory. C57Bl/60 mice and the rats were purchased from IFFA-Credo (L'Arbresle, France). Cells. Normal Iymphocytes were prepared from mouse spleen, thymus or Peyer's patches and from rat spleen, thymus, Peyer's patches, or axillary, inguinal and submandibular lymph nodes. The lymphoid organs were aseptically collected immediately after cervical dislocation, and ceils were dissociated by gentle teasing in cold Hank's balanced salt solution. Capsular debris and cellular agregates were eliminated by f i l t r a t i o n through nylon wool. Spleen cells were centrifuged in the cold (4°C) at 250xg for 10 rain. and the pellet was resuspended for 10 rain. in an ammonium chloride solution (8.3 g/l) to lyse erythrocytes. Spleen lymphocytes were then washed three times with IRPMX 1640 medium buffered with HEPES to pH 7.4 (Eurobio-Paris) and supplemented with penicillin (100 U/m]) and streptomycin (100 IJg/ml). Thymus, Peyer's patches and lymph nodes cells were washed twice in the same medium. Cells were adjusted to 1017 .ceJis/ml in this medium and preincubated for 30 rain. at 37°C in an atmosphere of 5% CO 2 in order to allow the cells to stabilise. Cells were then centrifuged and resuspended in fresh RPMI 1640 medium. Viability was measured by the F'osin-Y exclusion test and was always greater than 95 %. Mouse lymphoma ceil lines were grown in RPMI 1640 medium supplemented with penicillin, streptomycin, Lglutamine (2 raM) and 10 % fetal calf serum in a humidified atmosphere containing 5% CO2. A f t e r 3 days of culture the cells were washed twice in F
Vol. 40, No. 25, 1987
;,ymptlocyte cAMP Response
to DA
2387
Results Table [ shows cAMP response to DA (10-4M) and [SO (10-5M) in mouse and rat lymphocytes of various sources. DA induced a moderate but significant increase in cAMP levels in mouse spleen and thymus lymphocytes, but did not change cAMP content in Peyer's patches cells. The intensity of the increase by DA was the same in mouse spleen and thymus cells, and averaged approximately 50 %. DA-stimulated cAMP levels achieved 55 % and 28% of that induced by [SO in mouse spleen and thymus cells respectively. ISO also did not change cAMP accumulation in cells from mouse Peyer's patches. TABLE l Response to DA and ISO in mouse and rat lymphocytes of various sources. cAMP level Species
Mouse
Rat
(pmoles/107
cells)
I,}~nphoid tissue Basal
DA (IO-4M)
IS() (10-5M)
Spleen
7.77 + 0.47 (I-00) a
10.26 + 0.51 ~ (1_32)
19.47 + 1.36 ~ (2.31)
Thymus
4.39 + 0.35) (1-005
5.89 + 0.24 ~ (1.345
21.02 + I.£9 ~ (4.79)
Peyer's patches
6.53 _+ 0.65 (I.00)
6.14 + 0.43 (0.94)
5.70 + 0.28
7.72 + 0.57 ~
Spleen
( I .00) Thymus
Lymph nodes
Peyer's patches
(I_35)
6.62 + 0.53 (I .0t5 20.06 + 1.7t ~
(3.525
4.14 +_ 0.33 (1.005
4.60 + 0 17 (I_11)
8.01 + 0.56 ~ (1.94)
3.34 +_ 0.33
4.39 _+ 0.14 z
6.11 + 0.46 ~
(1.00)
(I .3t)
2.78 + 0.55
3.31 + 0 44
(1]005
(1.19]
(1.83) 3.05 + 0.57
(1.10)
Values are the mean + SD of 6-9 independent experiments. a) Mean ratio of cAMP level in presence of drug relative to basal cAMP level. * p < 0.05. In the rat, DA increased by approximately 30 % cAMP levels in Iymphoeytes from spleen and lymph nodes (table I). However, cAMP level in cells from rat thymus was not affected by DA whereas Peyer's patches lymphocytes were slightly (19 % increase) but not significantly sensitive to DA. Concentrations of cAMP in rat spleen and lymph nodes after stimulation by E)A were 38 % and -/2 %, respectively, of that produced by ISO. No change was observed in cAMP content of Peyer's patches cells in presence of ISO but unlike DA, ISO induced a 1.94-fold increase in cAMP level in rat thymocytes.
2388
Lymphocyte
cAMP
Response
to DA
Vol.
40, No.
25,
1987
The dose-response study of the c A M P a c c u m u l a t i o n in mouse and r a t spleen cells in presence o f increasing c o n c e n t r a t i o n s o f D A (table lI) showed s i g n i f i c a n t augmentations at 5 x 10-5 and 10-4M. No e f f e c t was observed at l o w e r c o n c e n t r a t i o n s in both species.
TABLE II. Dose-response e f f e c t of DA in mouse and r a t spleen lymphocytes. cAMP l e v e l
(pmoles/]07 cells)
DA (,~) Mouse 0
9.21
+ 0.76
(] .0o)a
Rat 5.93 + 0.03
(1_00)
10 -7
R.91 + 0.88 (0.97)
6. 17 + 0.25 (1_04)
10 -6
9.~7
6.2]
+ 0.17
(I .03) 5xi0 -6
10 -5
5xlO -5
10
+ 0.1@
(1.05)
9.54 + 0.97 (I .04)
6.4g _+ 0.31
10.52 + 1.40 (I.1~)
6.76 + 0.83 (1.14)
12.49 j o.95 ~ (1.36)
8.07
1 2 . 8 3 _~ 0 . 4 3 ~t (1 .39)
8 . 1 0 _+ 0 . 0 9 ~ ( l .37)
(1.09)
_+ 0 . ' ~ 9 ~t (I .36)
Values a r e t h e m e a n + SD o f 12 e x p e r i m e n t s . a) M e a n r a t i o o f cAM-P l e v e l in p r e s e n c e o f DA r e l a t i v e t o b a s a l c A M P level. * p < 0.05
As shown in t a b l e [II, among the 7 c e l l lines tested, only the Y A C - I T - l y m p h o m a cell line was sensitive to DA, w i t h a 8.50-fold increase in c A M P c o n t e n t of these cells as c o m p a r e d to baseline. D A - s t i m u l a t e d cAMP level in the Y A C - 1 cells achieved 42 % in comparison w i t h ISO. ISO also induced a p p r o x i m a t e l y a 2 - f o l d increase in cAMP levels in Sp2/0-Ag14 and BE104 cells, and about a 3 - f o l d increase in cAMP levels in 852AH9 cells. EL-4, C T L L - 2 and MOPC-315 cells did not respond to ISO. CTLL2 cells did not respond to CT. The responses of the o t h e r c e l l lines to CT varied f r o m m o d e r a t e (1.38-fold increase in MOPC-315 cells) to r e l a t i v e l y high (31.2-fold increase in Y A C - 1 cells). TH increased about 2 - f o l d c A M P c o n c e n t r a t i o n in a l l c e l l lines tested. D A - i n d u c e d increase in the cAMP c o n t e n t of Y A C - 1 ceils was dose-dependent (fig. 1). The threshold c o n c e n t r a t i o n was 5 x 10-6M which increased cAMP level by 33 %. The m a x i m a l e f f e c t was obtained at 10-4M, and DA c o n c e n t r a t i o n required to produce 50 % of m a x i m a l increase was about 5 x 10-5M.
Vol. 40, No. 25, 1987
Lymphocyte cAMP Response to DA
2389
Discussion DA (5 x 10-5 and 10-4M) provokes a moderate but consistent increase in cAMP content of normal mouse [ymphocytes from spleen and thymus. The intensity of the increase is of the same order for both organs, but the e f f e c t appears to be selective
T A B L E Ill Response to DA, lSO, CT and TH in mouse cell lines cAMP l e v e l (pmoles/107 c e l l s )
Cell llne a Basal
DA (10-4M)
ISO (IO-SM)
CT(100ng/ml)
TH(3xI0-3M)
531.i0+3.87 • (31.24)
29.07+i°16 ~ (I. 7 I)
YAC-I
17.00+0.23 (I .00) b
144.50+8.67 ~ (8.50)
348.16+17.41~ (20.48)
EL-4
11.82+1.30 (I .00)
10.99_+0.48 (0.93)
12.53+0.78 (i .06)
34.31+2.28 ~t 2~.87_+1.13 ~ (2 90) (2.02)
CTLL-2
14.84+1.01 (I.00)
15.58+0.37 (i.05)
]5.14_+0.03 (1.02)
14.69+0.73 (0 99)
24.47+1.11 ~ (1.65)
Sp2/O-Agl4
11.97+0.36 (I .00)
12.81+1.13 (1.07)
23,15+0.23 ~ (1.93)
37.23+3.69 ~ (3 II)
17.48_+0.87 • (1.46)
BEI04
6.77+~.78 (I.00)
7.06+0.68 (1.04)
12.15+0.5~ m (I.~0)
19.78+3.18 ~ (2 71)
17.52+1.95m (2.32)
852AH9
38.71+7.60 (I .00)
44.80+3.18 (1.16)
MOPC-315
48.24+3.87 (i .00)
56.20+3.93 (1.17)
101.72+11.70 m (2.63) 54.26_+1.44 (1.12)
214.84+28.54 ~ 66.77+8.37 m (5 55) (1.73) 66.57+5.37 ~ (I 38)
74.77+0.q2 ~ (I.SS)
Values are the mean + SD of 6-10 independent experiments. a) Strain origin and cell type of the cell lines are as follow: Y A C - I , A/Sn, Tlymphoma; EL-4, C57 Bl/6, thymoma; CTLL-2, C57 B1/6, T-cell clone; Sp2/0-Agl4, P3X63Ag8 x BALlS/c, non secreting myeloma; BE104, Sp2/O-Agl4 x BALB/c, monoelonal anti-B2-microglobulin antibody secreting hybridoma; 852AH99 Sp2/0Ag14 x BALlS/e, monoelonal anti-90 KDa a 1-microglobulin antibody secreting hybridoma; MOPC-315, BALlS/c, lgA secreting myeloma. The hybridomas BE 10b, and 852AH9 are obtained in our laboratory (C. Vincent) by fusion of Sp2/0-Agl4 myeloma cells and spleen cells of BALB/c mouse immunized with human thoracic duct Iymphocytes B2-microglobulin and purified al-microglobulin respectively. b) Mean ratio of cAMP level in presence of drug relative to basal cAMP level. • p < 0.05. since mouse lymphocytes from Peyer's patches do not respond to DA. The moderate rise in cAMP concentration in mouse sensitive [ymphocytes by DA contrasts with the results of Stepien et aL (25) who found a higher increase in cAMP level (100 - 200 %) and at lower doses of DA (10-6 and 10-5M) using young adult male Sprague-Dawley rat spleen
2390
Lymphocyte cAMP Response to DA
Vol. 40, No. 25, 1987
cells. These discrepancies w i t h our results cannot be a t t r i b u t e d to species d i f f e r e n c e s since in our hands spleen cells of r a t o f the same strain, the same sex and the same age as in the study of Stepien et aL (25), give the same m a g n i t u d e of response to DA as mouse spleen cells. D i f f e r e n c e s in the handling of cell suspensions and in the m e t h o d o l o g y of c A M P d e t e r m i n a t i o n can probably explain the d i f f e r e n t i n t e n s i t y of l y m p h o c y t e responses b e t w e e n our study and t h a t of Stepien e t a ] . (25). In this study we c o n f i r m the r a t h e r poor dose-response relationship of the O A induced increase in r a t spleen cells as r e p o r t e d by 5tepien e t a l . (25) who found 1D-6M and 10-SM D A c o n c e n t r a t i o n s to be equally e f f e c t i v e whereas l o w e r c o n c e n t r a t i o n s had no
,-~'~ 150 O O I-4
"~
lO0
,-4
0
~ 7
I 6
1 5
1 4
I 3
-Log DA concentration (M) FIG. l Dose-dependent increase in cAMP c o n t e n t of the Y A C - 1 cells by DA. e f f e c t . We e x t e n d this observation to n o r m a l mouse spleen cells. We also show t h a t r a t l y m p h nodes cells are sensitive to s t i m u l a t i o n by DA, whereas r a t lymphocytes f r o m Peyer's pstches are insensitive. In addition, and unlike mouse thymus cells, lymphocytes f r o m r a t thymus are insensitive to IDA. These results d e m o n s t r a t e an organ r e s t r i c t i o n not studied by others, in D A - i n d u c e d e|evation of c A M P levels in lymphoid cells, suggesting r e s t r i c t i o n to a c e l l p o p u l a t i o n possibly at c e r t a i n stages o f d e v e l o p m e n t . The responses of mouse and r a t n o r m a l I y m p h o c y t e s to ISO are always g r e a t e r than corresponding responses to DA. Mouse t h y m o c y t e s respond b e t t e r than spleen ceils to /SO c o n f i r m i n g the results of o t h e r authors (26, 27). On the c o n t r a r y , r a t thymus cell respond less w e l l than spleen cells but at the same e x t e n t as lymph nodes cells in this species. This result is in c o n t r a s t w i t h t h a t o f Makman (:~5) who found that basal and c a t e c h o l a m i n e - s t i m u l a t e d adenylate cyclase a c t i v i t y of r a t or mouse t h y m i c cells was g r e a t e r than t h a t of splenic or m e s e n t e r i c node cells in the same animal. I t must be pointed out t h a t this author used a d i f f e r e n t test system for cAMP d e t e r m i n a t i o n based on adenylate cyclase a c t i v i t y o f cell homogenates. C o n t r a d i c t o r y results have also been r e p o r t e d on the r e l a t i v e s e n s i t i v i t y of human l y m p h o c y t e s to ISO (29~ 34). Thus N i a u d e t e t aI. (54) found t h a t l y m p h o c y t e s f r o m thymus were more sensitive to ISO a c t i o n (8fold increase) than p e r i p h e r a l blood ]ymphoeytes (5-fold increase), whereas Van de Griend e t a | . (29) found t h a t human t h y m o c y t e s were not at all sensitive when I y m p h o c y t e s f r o m blood showed a 4 - f o l d increase in t h e i r i n t r a c e l | u ] a r content of cAMP in presence o f the drug.
Vol. 40, No. 25, 1987
Lymphocyte cAMP Response to DA
2391
The response of the Moloney leukemia virus (MLV)-induced lymphoma YAC-1 to DA is about 7 times greater than that of normal mouse or rat lymphoid cells. The response of YAC-1 cells to [SO and to CT are also the highest observed in comparison with the other cell lines. However, the YAC-1 cells do not seem to be sensitive to every known cAMPelevating agent, since histamine which is able to elevate cAMP levels in mouse lymphocytes by interacting with specific histamine H2-receptors (27) does not induce any significant effect in the YAC-1 cells under the same conditions as for DA, ISO, CT, and TH (data not shown). The YAC-1 cells are the sole among the 3 T-ceil lines to be sensitive to DA and ISO in our study. None of the 4 B-cell lines is sensitive to DA, whereas 3 of them respond appreciably to [SO. The unresponsiveness of the CTLL-2 cells to CT could be explained either by the lack of the specific membrane receptor of the toxin, the ganglioside GM1, or by a defect in the function of the adenylate cyclase system (35). YAC-1 cells have been shown to bear B-adrenergic receptors in binding studies (30) but so far their cAMP responses to catecholamines or to other drugs have not been examined. Since these cells are characterized by a progressive loss of H-2 antigens with concomitant increase of cell-surface antigens determined by MLV during serial in vitro passage (36), one could explain their sensitivity to cAMP-elevating agents by possible genetic alterations in components of the adenylate cyclase system. Nevertherless, cultured T- or B-lymphoma cell lines are thought to retain properties of their normal lymphocyte counterparts at various stages of development (37), and variations in the relative activities of the components of the adenylate cyclase system have ben observed to accompany differentiation in adipocytes (38) and in skeletal muscle cells (39). The relatively high response of the YAC-1 T-lymphoma cells to DA taken together with the lowest but organ-restricted response of mouse and rat normal non fractionated ]ymphocytes suggest that DA-sensitive adenylate cyclase might be limited to a small lymphocyte subset. Studies are currently in progress to confirm this hypothesis, and to determine the receptor specificity of the cAMP-elevating action of DA in lymphocytes. Since YAC-1 cells readily grow in culture, they could be used as a tool for pharmacological characterization of the DA-sensitive adenylate cyclase system.
Acknowledgments This study was supported by the MRES contract 508703.
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Pergamon Journals
CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE RESPONSE TO DOPAMINE IN MOUSE LYMPHOID CELLS AND CELL LINES E. Kouassi, Y.S. Li, M. Flacher and J.P. Revillard Laboratoire d'immunologie, INSERM U.80, CNRS UA 1177, UCBL, HSpital E. Herriot 690.37 Lyon Cedex 03, France (Received
in final form April 2, 1987)
Summary Cyclic adenosine 3',5'-monophosphate responses to dopamine and isoproterenol were studied in mouse and rat spleen, thymus, lymph nodes and Peyer's patches lymphocytes and in 7 mouse cell lines of T- and B-lymphoid derivation. The responses of normal cells to dopamine were moderate, of the same extent, but selective to spleen and thymus in mouse, and to spleen and lymph nodes in rat. The YAC-1 T Iymphorna cell line was sensitive to dopamine with a higher magnitude than normal lymphoid cells. Dopamine was less potent than isoproterenol in all celts, and whereas dopaminesensitive and isoproterenol-sensitive celts, or dopamine-insensitive and isoproterenol-insensitive cells were found, no cell type was dopamine-sensitive and isoproterenol-insensitive. Altogether, the.,~e results suggest that only a small subset of lymphocytes is susceptible to the cAMP-elevating action of dopamine. Lymphocyte responses may be regulated by catecho|amines released by nerve fibers which end adjacent to immune cells in thymus-dependent areas of lymphoid tissues (1). Many of the immunological effects of the 6-adrenergic catecholamines as well as those of numerous hormones, neurotransmitters and drugs, result from activation of the enzyme adenytate cyclase and the subsequently increased formation of intracellutar cyclic adenosine 3',5'-monophosphate (cAMP) which acts then as a "second messenger" (2-4). Dopamine (DA) can act on a-and 6-adrenergic receptors (5) and on two main types of specific receptors classified in respect to their association with adenylate cyclase activity (6): D-1 receptors which can cause an increase in adenylate cyclase activity, and 0-2 receptors which are not stimulatory [inked to the adenylate cyclase. Stimulation of D-2 receptors can in some cases even decrease adenylate cyclase activity (7). The existence of D-2 DA receptors have been postulated on mammalian lymphocytes (8) with B-cell selectivity (9) by using the binding of 3H-spiroperidol as a specific ligand. However, and although the number of binding sites in lymphocytes was affected in certain pathological conditions (10-16), a number of studies (15, 17-22) failed to confirm that 3H-spiroperido! binds to a specific DA receptor comparable to that of the brain (23) or peripheral (24) DA receptors. These studies suggested that the labelled ligand may be taken up by lysosomes. Conversely, Stepien et al. (25) showed that addition of DA at micromolar concentrations increased cAMP concentrations in the rat spleen cells, suggesting the presence of D-1 DA receptors on these cells although Shaskan et al. (22) could not demonstrate displaceable binding of a relatively selective D-1 DA receptor agonist 3H-2-amino-6,7-dihydroxy-l,2,%4-tetrahydronaphthalene (ADTN) on mouse thymus or spleen cells. 002/4-3025/87 $3.00 + .00 Copyright
(c) 1987 Pergamon Journals
Ltd.
2386
Lymphocyte cAMP Response to DA
Vol. 40, No. 25, 1987
In view of the important immunoregulatory role of cAMP in immunocytes, and the remarkable differences in the cAMP response to various hormones in subpopulations of ]ymphocytes related to their cell type and state of maturation and differentiation (2630), we investigated the cAMP response to DA in mouse Iymphocytes from spleen, thymus and Peyer's patches, and in various mouse T-and B-lymphoma cell lines. For comparative purposes, responses of rat spleen, thymus, lymph nodes and Peyer's patches cells~ and responses to the ~-adrenergie agonist isoproterenol (ISO) were studied. Materials and Methods Animals. Male and female 6-12 weeks old BALB/c or C57B1/63 mice and male 6-8 weeks old Sprague-Dawley rats served as normal cell donors. BALB/c mice were bred in our laboratory. C57Bl/60 mice and the rats were purchased from IFFA-Credo (L'Arbresle, France). Cells. Normal Iymphocytes were prepared from mouse spleen, thymus or Peyer's patches and from rat spleen, thymus, Peyer's patches, or axillary, inguinal and submandibular lymph nodes. The lymphoid organs were aseptically collected immediately after cervical dislocation, and ceils were dissociated by gentle teasing in cold Hank's balanced salt solution. Capsular debris and cellular agregates were eliminated by f i l t r a t i o n through nylon wool. Spleen cells were centrifuged in the cold (4°C) at 250xg for 10 rain. and the pellet was resuspended for 10 rain. in an ammonium chloride solution (8.3 g/l) to lyse erythrocytes. Spleen lymphocytes were then washed three times with IRPMX 1640 medium buffered with HEPES to pH 7.4 (Eurobio-Paris) and supplemented with penicillin (100 U/m]) and streptomycin (100 IJg/ml). Thymus, Peyer's patches and lymph nodes cells were washed twice in the same medium. Cells were adjusted to 1017 .ceJis/ml in this medium and preincubated for 30 rain. at 37°C in an atmosphere of 5% CO 2 in order to allow the cells to stabilise. Cells were then centrifuged and resuspended in fresh RPMI 1640 medium. Viability was measured by the F'osin-Y exclusion test and was always greater than 95 %. Mouse lymphoma ceil lines were grown in RPMI 1640 medium supplemented with penicillin, streptomycin, Lglutamine (2 raM) and 10 % fetal calf serum in a humidified atmosphere containing 5% CO2. A f t e r 3 days of culture the cells were washed twice in F
Vol. 40, No. 25, 1987
;,ymptlocyte cAMP Response
to DA
2387
Results Table [ shows cAMP response to DA (10-4M) and [SO (10-5M) in mouse and rat lymphocytes of various sources. DA induced a moderate but significant increase in cAMP levels in mouse spleen and thymus lymphocytes, but did not change cAMP content in Peyer's patches cells. The intensity of the increase by DA was the same in mouse spleen and thymus cells, and averaged approximately 50 %. DA-stimulated cAMP levels achieved 55 % and 28% of that induced by [SO in mouse spleen and thymus cells respectively. ISO also did not change cAMP accumulation in cells from mouse Peyer's patches. TABLE l Response to DA and ISO in mouse and rat lymphocytes of various sources. cAMP level Species
Mouse
Rat
(pmoles/107
cells)
I,}~nphoid tissue Basal
DA (IO-4M)
IS() (10-5M)
Spleen
7.77 + 0.47 (I-00) a
10.26 + 0.51 ~ (1_32)
19.47 + 1.36 ~ (2.31)
Thymus
4.39 + 0.35) (1-005
5.89 + 0.24 ~ (1.345
21.02 + I.£9 ~ (4.79)
Peyer's patches
6.53 _+ 0.65 (I.00)
6.14 + 0.43 (0.94)
5.70 + 0.28
7.72 + 0.57 ~
Spleen
( I .00) Thymus
Lymph nodes
Peyer's patches
(I_35)
6.62 + 0.53 (I .0t5 20.06 + 1.7t ~
(3.525
4.14 +_ 0.33 (1.005
4.60 + 0 17 (I_11)
8.01 + 0.56 ~ (1.94)
3.34 +_ 0.33
4.39 _+ 0.14 z
6.11 + 0.46 ~
(1.00)
(I .3t)
2.78 + 0.55
3.31 + 0 44
(1]005
(1.19]
(1.83) 3.05 + 0.57
(1.10)
Values are the mean + SD of 6-9 independent experiments. a) Mean ratio of cAMP level in presence of drug relative to basal cAMP level. * p < 0.05. In the rat, DA increased by approximately 30 % cAMP levels in Iymphoeytes from spleen and lymph nodes (table I). However, cAMP level in cells from rat thymus was not affected by DA whereas Peyer's patches lymphocytes were slightly (19 % increase) but not significantly sensitive to DA. Concentrations of cAMP in rat spleen and lymph nodes after stimulation by E)A were 38 % and -/2 %, respectively, of that produced by ISO. No change was observed in cAMP content of Peyer's patches cells in presence of ISO but unlike DA, ISO induced a 1.94-fold increase in cAMP level in rat thymocytes.
2388
Lymphocyte
cAMP
Response
to DA
Vol.
40, No.
25,
1987
The dose-response study of the c A M P a c c u m u l a t i o n in mouse and r a t spleen cells in presence o f increasing c o n c e n t r a t i o n s o f D A (table lI) showed s i g n i f i c a n t augmentations at 5 x 10-5 and 10-4M. No e f f e c t was observed at l o w e r c o n c e n t r a t i o n s in both species.
TABLE II. Dose-response e f f e c t of DA in mouse and r a t spleen lymphocytes. cAMP l e v e l
(pmoles/]07 cells)
DA (,~) Mouse 0
9.21
+ 0.76
(] .0o)a
Rat 5.93 + 0.03
(1_00)
10 -7
R.91 + 0.88 (0.97)
6. 17 + 0.25 (1_04)
10 -6
9.~7
6.2]
+ 0.17
(I .03) 5xi0 -6
10 -5
5xlO -5
10
+ 0.1@
(1.05)
9.54 + 0.97 (I .04)
6.4g _+ 0.31
10.52 + 1.40 (I.1~)
6.76 + 0.83 (1.14)
12.49 j o.95 ~ (1.36)
8.07
1 2 . 8 3 _~ 0 . 4 3 ~t (1 .39)
8 . 1 0 _+ 0 . 0 9 ~ ( l .37)
(1.09)
_+ 0 . ' ~ 9 ~t (I .36)
Values a r e t h e m e a n + SD o f 12 e x p e r i m e n t s . a) M e a n r a t i o o f cAM-P l e v e l in p r e s e n c e o f DA r e l a t i v e t o b a s a l c A M P level. * p < 0.05
As shown in t a b l e [II, among the 7 c e l l lines tested, only the Y A C - I T - l y m p h o m a cell line was sensitive to DA, w i t h a 8.50-fold increase in c A M P c o n t e n t of these cells as c o m p a r e d to baseline. D A - s t i m u l a t e d cAMP level in the Y A C - 1 cells achieved 42 % in comparison w i t h ISO. ISO also induced a p p r o x i m a t e l y a 2 - f o l d increase in cAMP levels in Sp2/0-Ag14 and BE104 cells, and about a 3 - f o l d increase in cAMP levels in 852AH9 cells. EL-4, C T L L - 2 and MOPC-315 cells did not respond to ISO. CTLL2 cells did not respond to CT. The responses of the o t h e r c e l l lines to CT varied f r o m m o d e r a t e (1.38-fold increase in MOPC-315 cells) to r e l a t i v e l y high (31.2-fold increase in Y A C - 1 cells). TH increased about 2 - f o l d c A M P c o n c e n t r a t i o n in a l l c e l l lines tested. D A - i n d u c e d increase in the cAMP c o n t e n t of Y A C - 1 ceils was dose-dependent (fig. 1). The threshold c o n c e n t r a t i o n was 5 x 10-6M which increased cAMP level by 33 %. The m a x i m a l e f f e c t was obtained at 10-4M, and DA c o n c e n t r a t i o n required to produce 50 % of m a x i m a l increase was about 5 x 10-5M.
Vol. 40, No. 25, 1987
Lymphocyte cAMP Response to DA
2389
Discussion DA (5 x 10-5 and 10-4M) provokes a moderate but consistent increase in cAMP content of normal mouse [ymphocytes from spleen and thymus. The intensity of the increase is of the same order for both organs, but the e f f e c t appears to be selective
T A B L E Ill Response to DA, lSO, CT and TH in mouse cell lines cAMP l e v e l (pmoles/107 c e l l s )
Cell llne a Basal
DA (10-4M)
ISO (IO-SM)
CT(100ng/ml)
TH(3xI0-3M)
531.i0+3.87 • (31.24)
29.07+i°16 ~ (I. 7 I)
YAC-I
17.00+0.23 (I .00) b
144.50+8.67 ~ (8.50)
348.16+17.41~ (20.48)
EL-4
11.82+1.30 (I .00)
10.99_+0.48 (0.93)
12.53+0.78 (i .06)
34.31+2.28 ~t 2~.87_+1.13 ~ (2 90) (2.02)
CTLL-2
14.84+1.01 (I.00)
15.58+0.37 (i.05)
]5.14_+0.03 (1.02)
14.69+0.73 (0 99)
24.47+1.11 ~ (1.65)
Sp2/O-Agl4
11.97+0.36 (I .00)
12.81+1.13 (1.07)
23,15+0.23 ~ (1.93)
37.23+3.69 ~ (3 II)
17.48_+0.87 • (1.46)
BEI04
6.77+~.78 (I.00)
7.06+0.68 (1.04)
12.15+0.5~ m (I.~0)
19.78+3.18 ~ (2 71)
17.52+1.95m (2.32)
852AH9
38.71+7.60 (I .00)
44.80+3.18 (1.16)
MOPC-315
48.24+3.87 (i .00)
56.20+3.93 (1.17)
101.72+11.70 m (2.63) 54.26_+1.44 (1.12)
214.84+28.54 ~ 66.77+8.37 m (5 55) (1.73) 66.57+5.37 ~ (I 38)
74.77+0.q2 ~ (I.SS)
Values are the mean + SD of 6-10 independent experiments. a) Strain origin and cell type of the cell lines are as follow: Y A C - I , A/Sn, Tlymphoma; EL-4, C57 Bl/6, thymoma; CTLL-2, C57 B1/6, T-cell clone; Sp2/0-Agl4, P3X63Ag8 x BALlS/c, non secreting myeloma; BE104, Sp2/O-Agl4 x BALB/c, monoelonal anti-B2-microglobulin antibody secreting hybridoma; 852AH99 Sp2/0Ag14 x BALlS/e, monoelonal anti-90 KDa a 1-microglobulin antibody secreting hybridoma; MOPC-315, BALlS/c, lgA secreting myeloma. The hybridomas BE 10b, and 852AH9 are obtained in our laboratory (C. Vincent) by fusion of Sp2/0-Agl4 myeloma cells and spleen cells of BALB/c mouse immunized with human thoracic duct Iymphocytes B2-microglobulin and purified al-microglobulin respectively. b) Mean ratio of cAMP level in presence of drug relative to basal cAMP level. • p < 0.05. since mouse lymphocytes from Peyer's patches do not respond to DA. The moderate rise in cAMP concentration in mouse sensitive [ymphocytes by DA contrasts with the results of Stepien et aL (25) who found a higher increase in cAMP level (100 - 200 %) and at lower doses of DA (10-6 and 10-5M) using young adult male Sprague-Dawley rat spleen
2390
Lymphocyte cAMP Response to DA
Vol. 40, No. 25, 1987
cells. These discrepancies w i t h our results cannot be a t t r i b u t e d to species d i f f e r e n c e s since in our hands spleen cells of r a t o f the same strain, the same sex and the same age as in the study of Stepien et aL (25), give the same m a g n i t u d e of response to DA as mouse spleen cells. D i f f e r e n c e s in the handling of cell suspensions and in the m e t h o d o l o g y of c A M P d e t e r m i n a t i o n can probably explain the d i f f e r e n t i n t e n s i t y of l y m p h o c y t e responses b e t w e e n our study and t h a t of Stepien e t a ] . (25). In this study we c o n f i r m the r a t h e r poor dose-response relationship of the O A induced increase in r a t spleen cells as r e p o r t e d by 5tepien e t a l . (25) who found 1D-6M and 10-SM D A c o n c e n t r a t i o n s to be equally e f f e c t i v e whereas l o w e r c o n c e n t r a t i o n s had no
,-~'~ 150 O O I-4
"~
lO0
,-4
0
~ 7
I 6
1 5
1 4
I 3
-Log DA concentration (M) FIG. l Dose-dependent increase in cAMP c o n t e n t of the Y A C - 1 cells by DA. e f f e c t . We e x t e n d this observation to n o r m a l mouse spleen cells. We also show t h a t r a t l y m p h nodes cells are sensitive to s t i m u l a t i o n by DA, whereas r a t lymphocytes f r o m Peyer's pstches are insensitive. In addition, and unlike mouse thymus cells, lymphocytes f r o m r a t thymus are insensitive to IDA. These results d e m o n s t r a t e an organ r e s t r i c t i o n not studied by others, in D A - i n d u c e d e|evation of c A M P levels in lymphoid cells, suggesting r e s t r i c t i o n to a c e l l p o p u l a t i o n possibly at c e r t a i n stages o f d e v e l o p m e n t . The responses of mouse and r a t n o r m a l I y m p h o c y t e s to ISO are always g r e a t e r than corresponding responses to DA. Mouse t h y m o c y t e s respond b e t t e r than spleen ceils to /SO c o n f i r m i n g the results of o t h e r authors (26, 27). On the c o n t r a r y , r a t thymus cell respond less w e l l than spleen cells but at the same e x t e n t as lymph nodes cells in this species. This result is in c o n t r a s t w i t h t h a t o f Makman (:~5) who found that basal and c a t e c h o l a m i n e - s t i m u l a t e d adenylate cyclase a c t i v i t y of r a t or mouse t h y m i c cells was g r e a t e r than t h a t of splenic or m e s e n t e r i c node cells in the same animal. I t must be pointed out t h a t this author used a d i f f e r e n t test system for cAMP d e t e r m i n a t i o n based on adenylate cyclase a c t i v i t y o f cell homogenates. C o n t r a d i c t o r y results have also been r e p o r t e d on the r e l a t i v e s e n s i t i v i t y of human l y m p h o c y t e s to ISO (29~ 34). Thus N i a u d e t e t aI. (54) found t h a t l y m p h o c y t e s f r o m thymus were more sensitive to ISO a c t i o n (8fold increase) than p e r i p h e r a l blood ]ymphoeytes (5-fold increase), whereas Van de Griend e t a | . (29) found t h a t human t h y m o c y t e s were not at all sensitive when I y m p h o c y t e s f r o m blood showed a 4 - f o l d increase in t h e i r i n t r a c e l | u ] a r content of cAMP in presence o f the drug.
Vol. 40, No. 25, 1987
Lymphocyte cAMP Response to DA
2391
The response of the Moloney leukemia virus (MLV)-induced lymphoma YAC-1 to DA is about 7 times greater than that of normal mouse or rat lymphoid cells. The response of YAC-1 cells to [SO and to CT are also the highest observed in comparison with the other cell lines. However, the YAC-1 cells do not seem to be sensitive to every known cAMPelevating agent, since histamine which is able to elevate cAMP levels in mouse lymphocytes by interacting with specific histamine H2-receptors (27) does not induce any significant effect in the YAC-1 cells under the same conditions as for DA, ISO, CT, and TH (data not shown). The YAC-1 cells are the sole among the 3 T-ceil lines to be sensitive to DA and ISO in our study. None of the 4 B-cell lines is sensitive to DA, whereas 3 of them respond appreciably to [SO. The unresponsiveness of the CTLL-2 cells to CT could be explained either by the lack of the specific membrane receptor of the toxin, the ganglioside GM1, or by a defect in the function of the adenylate cyclase system (35). YAC-1 cells have been shown to bear B-adrenergic receptors in binding studies (30) but so far their cAMP responses to catecholamines or to other drugs have not been examined. Since these cells are characterized by a progressive loss of H-2 antigens with concomitant increase of cell-surface antigens determined by MLV during serial in vitro passage (36), one could explain their sensitivity to cAMP-elevating agents by possible genetic alterations in components of the adenylate cyclase system. Nevertherless, cultured T- or B-lymphoma cell lines are thought to retain properties of their normal lymphocyte counterparts at various stages of development (37), and variations in the relative activities of the components of the adenylate cyclase system have ben observed to accompany differentiation in adipocytes (38) and in skeletal muscle cells (39). The relatively high response of the YAC-1 T-lymphoma cells to DA taken together with the lowest but organ-restricted response of mouse and rat normal non fractionated ]ymphocytes suggest that DA-sensitive adenylate cyclase might be limited to a small lymphocyte subset. Studies are currently in progress to confirm this hypothesis, and to determine the receptor specificity of the cAMP-elevating action of DA in lymphocytes. Since YAC-1 cells readily grow in culture, they could be used as a tool for pharmacological characterization of the DA-sensitive adenylate cyclase system.
Acknowledgments This study was supported by the MRES contract 508703.
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Vol. 40, No. 25, ]987
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