Thymic hormones and prostaglandins. I. Lack of stimulation of prostaglandin production by thymic hormones

PROSTAGLANDINS

THYMIC HORMONES AND PROSTAGLANDINS. I. LACK OF STIMULATION OF PROSTAGLANDIN PRODUCTION BY TBYMIC HORMONES F. Homo-Delarche, J-F. Bach, and M. Dardenne INSERM U 25 and

161 rue

de SBvree,

CNRS UA 122, 75743 Paris

H8pital Necker, Cedex 15, France

ABSTRACT Prostaglandins (PGs) have been implicated as possible mediators of the biological activity of thymic hormones. It has been shown that type E-PGs are able to mimic the action of several thymic hormones and that indomethacin prevents in vivo or in vitro the appearance of Thy-l+ antigen induced by some of these factors. We thus investigated a possible role for PGs in the mechanism of action of different thymic extracts and peptides. Attempts to modulate prostaglandin production showed that neither thymosin fraction 5 (0.01 - 100 pg/ml), nor thymosin a 1 (l-10 l.tg/ml), thymulin (0.001-100 ng/ml), thymopoietin II (10 - 1000 ng/ml) or TP5 (10 - 1000 ng/ml) affect PGE2, 6-ketoPGFl a, PGF2 a and TXB2 production by spleen cells from control and thymectomized mice. These results do not support the hypothesis that prostaglandins could act as mediators of thymic hormones. INTRODUCTION The effects of thymic hormones (TH) on T-cell differentiation are well documented (l-5). T-cell differentiation has also been shown to be induced by cyclic AMP (cAMP)and substances such as P-adrenergic agents and type E-prostaglandins, which led to the conclusion that thymic hormones may act via the adenylate cyclase system (1,2, 6-8). However, evidence of CAMP as the second messenger for thymic hormones still remains a matter of debate (3-7, 9-l 1). Nevertheless, the implication of PGs as possible mediators of the biological activity of thymic hormones persists on the basis of several arguments : 1) type EPGs have been shown to mimic the action of several TH, since they modulate in vitro the expression of T-cell differentiation antigens (such as Thy-l) on immature T cell precursors (1,6,8). Moreover, a long-acting synthetic analog of prostaglandin E2 (PGE2), 16, 16-

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dimethyl-PGE2 ester is able to increase some of the reduced functions in adult thymectomized (TX) mice (12) ; 2) indomethacin prevents, in vivo and in vitro, the appearance of Thy-l antigen induced by thymulin and thymosin fraction 5 (TF5), which would suggest that thymic hormone action would require prostaglandin synthesis (13,14) ; 3) in this regard, two brief reports demonstrate that PGE2 production is stimulated by thymulin on the one hand, and TF5 as well as thymosin al, on the other (15,16). Despite these data, we decided to reinvestigate the role of PGs in the biological activity of thymic hormones for several reasons : 1) in the conditions in which the activity of various TH have been tested, i.e., the induction of T-cell markers on, for example, splenocytes from thymectomized mice used as targets (16), other PGs than PGE2, which is relatively unstable, may be produced ; 2) indomethacin has been shown to exert other effects than inhibition of cyclooxygenase, such as inhibition of phosphodiesterase or protein kinase (17,lQ thus allowing possible misinterpretation of the data ; and 3) finally, the effects of different thymic hormones which are represented by thymic extracts, such as TF5, or pure thymic peptides, such as thymosin al, thymulin, thymopoietin II and TP5, have never been compared on PG production by thymocytes or by spleen cells, isolated either from normal or from thymectomized mice.

MATERIALS

AND METHODS

Animals : mice were 4-6 week old male and female C57BL/6, bred in our animal facilities under SPF conditions. Adult thymectomy was performed under ether anaesthesia by suction with a Pasteur pipet. Sham thymectomized (TX) mice as well as normal mice were used as controls. Reagents : Hanks’ balanced salt solution (HBSS) was obtained from Eurobio (Paris, France). Thymulin was synthesized by Pierre Lefrancier (Choay Institute, Paris, France). TP5 and thymopoietin II as well as thymosin Fraction 5 were kindly donated by Gideon Goldstein (Raritan, NJ,USA) and Allan Goldstein (Washington, DC, USA), respectively. Thymosin al and indomethacin were bought from Sigma (St-Louis, MO,USA), azathioprine from Wellcome (Dartford, UK) and nonradioactive prostaglandins from Seragen Inc. (Boston, MA, USA). (> lOOCi/mmol) were purchased from Tritiated prostaglandins

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Amersham Center (UK). Prostaglandin antisera were obtained from Biosys (Compiegne, France). All organic solvants used in the extraction of prostanoid were obtained from Baker (Deventer, Holland). Indomethacin was dissolved in ethanol to make IO-TM stock solution, and the final concentration in the sample was equal to 0.01% and did not modify cell secretion. Cell preparation : spleen cells and thymocyte suspensions were obtained in HBSS, washed twice and adjusted to a concentration of 40 x 106 cells/ml. Aliquots of lOOp1 of the cell suspension were incubated for 75 min at 37°C in an incubator with various dilutions of the drug to be tested, in a final volume of 470~1. After incubation, aliquots of cell-free supernatant were rapidly stored at -20°C before extraction for prostaglandin measurement. Extraction of the prostaglandin fraction and radioimmunoassay (RIA) for PGs : aliquots of the culture medium (250~1) were mixed with acetone (1:2 v/v) to precipitate proteins. After centrifugation, supematants were treated with 2 volumes of hexane. After acidification to pH 3.5 with 70% citric acid, the inferior phases were mixed with 2 volumes of chloroform. Following agitation at 4°C overnight, the inferior phases were evaporated under nitrogen. Yield was assessed using tritiated prostaglandins. For RIA, corrections were made according the recovery ratio of each eicosanoid. After extraction, the PG containing fractions were suspended in 0.1 M sodium chloride/phosphate buffer pH 7.4, containing 0.1% gelatin. The radioimmunoassays for PGE2 ,6-keto PGFla, PGF2a and TXB2 were performed according to the method of Dray et al. (19) as described elsewhere (20). Statistical analysis : statistical using the Student’s t-test.

significances

were

determined

by

RESULTS 1. Spontaneous prostanoid spleen cells.

production

by unseparated

mouse

Figure 1 shows the kinetics of spontaneous PGE2, PGF2a, keto-PGFla and TXB2 productions over a 75 min period incubation. In these conditions, the production of each type

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6of of

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prostanoid prostanoid population Table 1.

increased slightly as a function of time. The pattern of whole spleen production by an unstimulated, cell from control mice, as measured by RIA, is shown in

0 0

15

30

45

60

75

Time (min)

Figure 1 : Kinetics of prostaglandin production by unseparated spleen cells from control mice. Cells (4 x 106/470pl HBSS) were incubated for 75 min. At various intervals, the supernatants were removed, extracted and the secretions of PGE2 (o), PGF2a (A), 6-keto- PGFl a (0) and TXB2 ( 0 ) were measured by specific RIA. Each value represents the mean of three individual determinations in a typical experiment.

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Although the total levels of PG measured may vary among experiments, the secretion of the different types of PG measured is relatively constant when expressed as a percentage of total secretion. The two major compounds released into the medium were 6-keto-PGFla (36.6 I!Z 13.6%) and TXB2 (29.7 f 9.1%), whereas PGE2 and PGF2a represented 15.7 ?I 6.2% and 18.1 f 5.6%. These results are in with those obtained using high pressure liquid agreement chromatography (HPLC) after cell labeling with 14C-arachidonic acid, where we principally detected 6-keto-PGFla and TXB2 and only small amounts of PGE2 and PGF2a (results not shown). No significant difference was observed between prostanoid production by spleen cells from control, sham thymectomized and thymectomized animals, when measured in comparative experiments. TABLE

1 : Pattern cells

Total secretion of PG (pg/4 x 106 cells)

Range Mean SD

1598-6068 3380 + 1139

of prostanoid

PGE2

production

PGE2a

by mouse

6-keto-PGFl

a

spleen

TXB2

(% of total secretion)

10-25.8 15.7 + 6.2

1 l-27 18.1 + 5.6

21-55 36.6 + 13.6

15-43 29.7 f 9.1

4 x 106 unseparated spleen cells were incubated for 75 min as described in Figure 1. The secretion of PGE2, PGF2a, 6-keto-PGFla and TXB2 was measured by radioimmunoassay in 14-16 experiments. The proportion of each type of prostaglandin was expressed as percentage of total prostaglandin production and resulted from triplicate determination in each experiment.

2. Effect of increasing concentrations prostanoid production by unseparated control and TX mice.

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of thymulin on spleen cells from

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Figure 2 shows the effect of different thymulin concentrations (from 0.001 to 100 t-&ml), which are currently used in the biological assay conditions of the hormone. At none of the concentrations tested, thymulin was able to modulate the production of the different types of prostanoid investigated, in either normal or thymectomized animals.

Control

0

PGEZ

--“8

8 0

PGF2,,

-1

-

EIl

Thymectonwed

6 keto PGF,,,

g-es

.g-ss

0

0

TX62 I

I

G-c20 -

z

-0

Thymukn concentration (ng/ml)

Figure 2 : Effect of increasing concentrations of thymulin (0.001-100 ng/ml) on PGE2, PGF2a, 6-keto-PGFla and TXB2 production by unseparated spleen cells from control (0) and TX mice ( q ), in the same experimental conditions as described in Figure 1. Immunoreactive production of PGs in the absence of reagent (control) is taken as 100% and the results obtained in the presence of thymulin are expressed as percentages of these control values. Each value represents the mean of triplicate determinations in 5 experiments.

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TABLE 2 : Effect of different thymic hormones (TH) on PGE2, 6-keto-PGFla production by unseparated spleen cells from normal and TX mice.

PGE2 Thymic hormones Thymosm Fraction 5 @g/ml) 0.01 10 100 Thymosin al @g/ml) : 10 Thymopoietin II @g/ml) 10 100 1000 TP5 (ng/ml) 10 100 1000

Control

and TXB2

Prostaglandinproduction(% of control values) 6-keto-PGFla TXB2 TX Cmltrol TX ConUOl

TX

119+1x 1081k16 99+17 109 * 30

101*18 107-t24 101 * 21 921t27

95 k 7 97 * 4 96ill 97 + 8

113+23 10x*2 116f 14

106+5 108 i 13 96k 18

108f 8 103 It 10 99f 16

lob+

97fl2 102i4

8bk

13 78+14

87f18 107f23 97k12

102f 13 120+7 119*27

101*4 101+4 126 f 28

107k 6 105* 19 99* 17

9bf7 95f12 108 f 17

90f 19 85f18 86f 10

102f 13 109f21 117f 14

109k 16 96 +5 lO6+ II

98 f 23 102k 11 91 k 14

99f5 99+8 105*5

109f 13 96+ 15 94f9 102 + 12 12

94f3 98 f 32 87+12

102f36 93 + 28 112*20 102*2 85+4

86+ 18 98f32 119k21

105f21 115f17 102f 13 98* 18

107 * 10 95f 12 78f9

hmmmoreactwe PG productmn m Ihe abscncc of reagents (control) is taken as 100% and Ihe nx.ul~~obtained in tie presence of TH are expressed as percentages of these control values. Each value representsIhe mean of triplicate determinations m 3 expaimenls for TP5. lhymosm frackm 5. thymosin al and thymopoxtm II.

3. Comparison of the effect of different thymic hormones on prostanoid production by unseparated spleen cells from control and TX mice and on unfractionated thymocyte population. We then compared the effect of different thymic hormones or extracts or peptides, i.e., thymosin fraction 5, (0.01 - 100 pg/ml), thymosin a 1 (1 - 10 pg/ml), thymopoietin II (10 - 1000 ng/ml) and TP5 (10 - 1000 r&ml). In no case, these hormones, tested in a wide range of concentrations, were able to modulate prostanoid production by unseparated spleen cells isolated from either control or thymectomized mice (Table 2). Moreover, in unfractionated thymocyte population, we were only able to detect a small spontaneous PGE2 production (91 f 47 pg/4 x 106 cells, n = 7). The addition of the

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different thymic peptides or extracts, thymosin fraction 5, thymosin a 1, thymulin, thymopoietin II and TP5 had no effect on PGE2 production (Table 3). TABLE

3 : Effect of different thymic hormones on PGE2

production tion.

by unfractionated

thymocyte popula-

PGE2*production (% of control values)

Thymuline (rig/ml) 0.001 0.1 100 Thymosin fraction 5 @g/ml) 0.01 1 10 100 Thymosin al @g/ml) 1 5 10 Thymopoietin (@ml) 10 100 1000 TP5 (r&ml) 10 100 1000

112.6 + 20 113.9 * 34.4 102.9 f 1.3 120 102 104.4 81.9

+ 11.6 ? 23 * 22 f 8.8

81.7 ? 23 80 + 12.5 81.5 f 15

85.6 f 3.7 108.4 + 49 146 f 51 92.3 IL11.3 85.6 k 10.1 100.1 f 16.5

Immunoreactive PG production in the absence of reagents (91 f 47 pg/ 4 x 106 thymocytes, n = 7) is taken as 100% and the results obtained in the presence of TH are expressed as percentages of these control values. Each value represents the mean of triplicate determination in 3 experiments for each TH.

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on prostanoid production 4. Effects of indomethacin unseparated spleen cells from control and TX mice.

by

We tested the effect of lo-6M indomethacin on the spontaneous production of PGE2, PGF2a, 6-keto-PGFla and TXB2. As expected, indomethacin induced a significant (p < 0.001) and similar (- 60%) inhibition of the production of all types of PGs tested, by unseparated spleen cells from either control or thymectomized mice over a 7.5 min incubation (Table 4). TABLE

4 : Effect of indomethacin

(IO-6M) on prostanoid production by unseparated cells from normal and TX mice

PG production (% of control values)

Control mice

TX mice

PGE2 PGF2a 6-keto-PGFl TXB2

37.7 41.3 44.6 39.9

38.2 42.2 33.5 37

a

f + + f

8 9.4 9.6 14.8

f 8.4 k 12 f 13.4 fll

Immunoreactive PG production in the absence of reagents (control) is taken as 100% ; the results in the presence of indomethacin are expressed as percentages of these control values and result from triplicate determinations in 8-l 2 experiments. Statistical significances were determined by using the Student’s t-test : p < 0.001 in all groups.

DISCUSSION role for PGs in the mechanism of in we showed that spleen cells isolated from either control or TX mice spontaneously produced different types of PGs, as first assessed by identification using 14C arachidonic acid labeling and then radioimmunoassay measurements. 6-keto-PGFl a and TXB2 were the major compounds released, representing 40% and 30% Reinvestigating

a possible

vitro action of thymic hormones,

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of the total secretion respectively, whereas PGE2 and PGF2a represented around 15%. However, it should be emphasized that PGE2 production is systematically underestimated, due to its instability in aqueous medium (21). Spleen cells are a heterogeneous population composed mainly of T and B lymphocytes and a small proportion of macrophages. However, among these different populations, macrophages are the potential producers of PGs. This was verified after separation of spleen cells by adherence (results not shown). Attempts to modulate PG production by different thymic hormones show that neither thymulin nor thymosin fraction 5, thymosin al, thymopoietin II or TPS affect PG production by spleen cells from control or TX mice nor do they have any effect on small spontaneous PGE2 production by thymic cells.With regard to the thymic population, as well as spleen cells, it is more or less directly assumed that the contaminating nonlymphoid cell population (i.e., macrophages and accessory cells) is responsible for the prostanoid production (22-24). We found, as others (22, 24), a very low immunoreactive PGE2 production and no significant detectable amounts of radioactive PG after HPLC analysis (results not shown). Our present results do not agree with the reports showing that TF5 and thymosin al are able to induce an early release of PGE2 by thymocytes as well as spleen cells collected from ATx mice (16,25), but agree with the finding that thymulin is unable to stimulate PGE2 production by unfractionated thymocytes (26). The few available data concerning the action of thymic hormones on PG production are somewhat contradictory , since thymulin, TP-5 and thymosin fraction 5 do not stimulate PG production by guinea-pig parenchymal lung tissue (as assessed by radioimmunoassay (27)), whereas thymulin was shown to induce PGE2 production by human peripheral mononuclear cells as measured by gas chromatograph-mass spectometry, the latter results being more variable when using only peripheral nonadherent cells (15). The reasons for these discrepancies are unknown, but they may involve the use of different target tissue or cells and/or different techniques. Nevertheless, it should be emphasized that the present work is the first attempt to compare the effects of different thymic hormones on PG production by whole thymocyte or spleen cell suspensions isolated from normal or TX mice. The lack of in vitro PG stimulation by the different thymic hormones does not exclude that either “exogenous” PGs exert a direct role on the expression of Thy-l antigen as previously demonstrated (1,6,8) or even that endogeneously produced PGs play an indirect role

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in the expression of this membrane antigen as well as in the biological activity of TH and in particular in the biological assay for thymulin (4): we showed that, in the conditions of this assay, endogeneous PG production was inhibited by approximately 60%. Nevertheless, if we are unable to demonstrate an effect of thymic hormones on prostanoid synthesis, we cannot exclude that thymic hormones and prostaglandins act synergistically both in vivo and in vitro. In order to demonstrate a synergistic effect of thymic hormones and PGs in vitro, we are studying whether : 1) the decreased biological effect of thymulin observed in the presence of cyclooxygenase inhibitors, can be reversed by concomittantly adding PGs ; 2) the effect of endogeneously produced PGs in this system on thymic hormone action can be abolished by the addition of specific anti-prostaglandin antibodies ; 3) the simultaneous addition of thymic hormones and PGs, at concentrations which themselves have no effect, is active in the assay. ACNOWLEDGMENTS The authors wish to thank Marie-Claude Gagnerault and Josiane Simon for their skillful1 technical assistance, Catherine Slama and Doreen Broneer for their help in the preparation of the manuscript. REFERENCES 1.

SCHEID, M.P., G. GOLDSTEIN andE.A. BOYSE. The generation and regulation of lymphocyte populations. Evidence from differentiative induction system in vitro. J. Exp. Med. 147 : 1727,1978.

2.

SCHEID, M.P., G. GOLDSTEIN, U. HAMMERLING and E.A. BOYSE. Lymphocyte differentiation from precursor cells in vitro. Ann. N.Y. Acad. Sci. 249 : 53 1, 1975.

3.

NAYLOR, P.H. and A.L. GOLDSTEIN. Thymosin : Cyclic nucleotides and T cell differentiation. Life Science 25 : 301, 1979.

4.

BACH, J.F. Thymulin 3 : 133, 1983.

5.

OATES, K.K. and A.L. GOLDSTEIN. Thymosins

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(FI’S-Zn). Clinics in Immunol

& Allergy

: hormones

of the

193

PROSTAGLANDINS

thymus. Trends in Pharmacol. Sci. 5. : 347, 1984. 6.

BACH, J.F. and M.A. BACH. Studies on thymus products. VI. The effects of cyclic nucleotides and prostaglandins on rosetteforming cells. Interactions with thymic factors. Eur. J. Immunol. 3 : 778, 1973.

7.

KOOK, A.I. and N. TRAININ. Hormone-like activity of a thymus humoral factor on the induction of immune competence in lymphoid cells. J. Exp. Med. 139 : 193, 1974.

8.

SINGH, U. and J.J.T. OWEN. Studies on the maturation of thymus stem cells. The effects of catecholamines, histamine and peptide hormones on the expression of T cell alloantigens. Eur. J. Immunol. 6 : 59, 1976.

9.

BACH, M.A. Sera from both normal and thymectomized animals produce elevations of cyclic AMP in thymocytes. Cell Immunol. 33 : 224, 1977.

10.

NAYLOR,P.H., C.E. CAMP, A.C. PHILIPS, G.B. THURMANand A.GOLDSTEIN.Effect of thymosin and lipopolysaccharide on murine lymphocyte cyclic AMP. J. Immunol. Methods. 20 : 143, 1978.

11.

SUSHINE,G.H., R.S. BASH, R.G. COFFEY, K.W. COHEN, G. GOLDSTEINand J.W. HADDEN.Thymopoietin enhances the allogeneic response and cyclic GMP levels of mouse peripheral, thymus-derived lymphocytes. J. Immunol. 120 : 1594, 1978.

12.

GARACI, E., G.C. RINALDI,V. DELGOBBO,C. FAVALLI, M.G. SANTOROand B. JAFFE. A synthetic analog of prostaglandin E2 is able to induce in vivo theta antigen on spleen cells of adult thymectomized mice. Cell. Immunol. 62 : 8, 1981.

13.

BACH, M.A., C. FOURNIERand J.F. BACH. Regulation of thetaantigen expression by agents altering cyclic AMP levels and by thymic factor. Ann. N.Y. Acad. Sci. 249 : 316, 1975.

14.

RINALDI-GARACI,C., V. DEL GOBBO,C. FAVALLI,E. GARACI, F. BISTONI and B.N. JAFFE. Induction of serum thymic-like

194

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PROSTAGLANDINS

activity in adult thymectomized mice by a synthetic analog of PGE2. Cell.Immunol. 72 : 97, 1982. 15.

GUALDE, N., M. RIGAUDand J.F. BACH. Stimulation of prostaglandin synthesis by the serum factor (ITS). Cell. Immunol. 70 : 362,1982.

16.

RINALDIGARACI,C., C. FAVALLI,V. DEL GOBBO,E. GARACI and B.M. JAFFE. Is thymosin action mediated by prostaglandin release ? Science -. 220 . 1163, 1983,

17.

FLOWER,

18.

KANTOR,H. and M. HAMPTON.Indomethacin in submicromolar concentrations inhibits cyclic AMP-dependent protein kinase. Nature -* 276 * 841, 1978.

19.

DRAY, F., B. CHARBONNELand J. MACLOUF. Radioimmunoassay of prostaglandins F2, El, and E2 in human plasma. Eur. J. Clin. Invest. 5, 3 11, 1975.

20.

HOMO-DELARCHE,F., D. DUVAL, and M. PAPIERNIK. Prostaglandin production by phagocytic cells of the mouse thymic reticulum in culture and its modulation by indomethacin and corticosteroids. J. Immunol. -1 135 * 506, 1985.

21.

DURANT,S., D. DUVALand F. HOMO-DELARCHJZ. Mouse embryo fibroblasts in culture : characteristics of arachidonic acid metabolism during early passages. Prost. Leuk. & Med. 32 : 129, 1988.

22.

TOMAR, R.H., T.L. DARROWand P.A. JOHN. Response to and production by murine thymus, spleen, bone marrow, and lymph node cells. Cell. Immunol. 60 : 335, 1981.

23.

SHIPMAN,P.M., R.R. SCHMIDTand K.P. CHEPENIK.Relation between arachidonic acid metabolism and development of thymocytes in fetal thymic organ cultures. J. Immunol. 140 : 2714,1988.

AUGUST

R. Drugs which inhibit prostaglandin synthesis. Pharmacol. Rev. 26 : 33, 1974.

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24.

DUVAL, D. Effect of dexamethasone on arachidonate metabolism in isolated mouse thymocytes. Prost. Leuk. & Med (in press).

25.

GARACI, E., C. FAVALLIand C. RINALDI-GARACI.Interaction of thymic hormones with prostaglandins. In : Thymic hormones and lymphokines (A.L. Goldstein Ed.) Plenum Press New York and London, 1984, p. 413.

26.

RINALDI-GARACI,C., T. JEZZI, M. BALDASSARE,M. DARDENNE,J.F. BACH and E. GARACI. Effect of thymulin on intracellular cyclic nucleotides and prostaglandin E2 in peanut agglutinin-fractionated thymocytes. Eur. J. Immunol. 15 : 548, 1985.

27.

STEEL, L.K., J.F. WEISS and G.N. CATRAVAS.In vitro prostaglandin release from guinea pig parenchymal lung tissue is not stimulated by thymic peptides. Int. J. Immunopharmacol. 6 : 75, 1984. Editor:

196

J. Roberts

II

Received:

l-30-89

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Accepted:

6-7-89

1989 VOL. 38 NO. 2