The role of prostaglandins and thromboxane A2 on mitogen-induced peripheral blood mononuclear cell proliferation

Prog. Lipid Res. Vol. 25, pp. 401--404, 1986 Printed in Great Britain. All rights reserved

0163-7827/86]$0.00 + 0.50 © 1986 Pergamon Journals Ltd

THE ROLE OF PROSTAGLANDINS AND THROMBOXANE A2 ON MITOGEN-INDUCED PERIPHERAL BLOOD MONONUCLEAR CELL PROLIFERATION W. AW~A,* I~rrH HILLIER* and DAVID JONES~ *Clinical Pharmacology and tExperimental Pathology Medical Faculty, University of Southampton, Soulhampton S09 3TU, U.K.

INTRODUCTION Data are accumulating that describe a place for the eicosanoids in immunological regulation. 2"3 It is suggested that certain eicosanoids are mediators or modulators of immune and inflammatory reactions and that the eicosanoid modulation may be defective in the immunological component of some clinical disorders) Because a large number of cyclooxygenase and lipoxygenase derived arachidonic acid metabolites can be synthesized by immunocompetent cells, the definition of their role is complex. Goodwin et al. 4 showed that PGE~ inhibited mitogen-induced lymphocyte proliferation (LP) and subsequently Kelly et al. 7 suggested a similar role for TxA~ which is the major cyclooxygenase product in human peripheral blood mononuclear cells (PBMNC). The latter conclusion was derived solely from the use of TxA e synthesis inhibitors and was subsequently refuted by another investigation. 5 In the last few years, investigations into the effects of the eicosanoids on LP resulted in many inconsistencies and unexplained variability; 2 thus, PGE2 inhibitory potency increases as the amount of mitogen-induced proliferation decreases. Also, indomethacin does not consistently increase mitogen-induced cell proliferation but is more effective with sub-optimal concentrations of phytohaemagglutinin (PHA); 4 this throws doubt on the importance of cyclooxygenase products of arachidonic acid in modulating LP. We felt that inconsistencies could have arisen because of the lack of attention paid to endogenous PG synthesis in control cultures and in the presence of blocking drugs. We have reassessed the way in which PGE2 and TxA 2 are synthesized in PHA-stimulated PBMNC and the role of TxA2 and PGE2 using the selective TxA2 synthesis inhibitor (UK37248), a TxA 2 receptor stimulant (U46619) and TxA2 receptor antagonist (EP045).

MATERIALS AND METHODS Heparinized peripheral blood was obtained from healthy donors not receiving medication. Mononuclear cells were isolated and proliferation in response to PHA using 3H-thymidine incorporation assessed according to the method of Akbar and Jones. I PGF_~ and TxA2 were assayed according to the method of Hillier et al. ~ with one modification. Eicosanoids were purified by column chromatography using octadecasilylsilica columns before measurement of the eicosanoids by radioimmunoassay. With all drugs used, the cell viability after 48 hr incubation was more than 95% as measured by trypan blue exclusion. Control cultures were always carried out in the presence of the diluents used for preparing the drug additions. In the experiments carried out to measure eicosanoid synthesis, 106 cells in ! ml of culture medium were incubated for up to 48 hr. Cells were centrifuged and the supernatant assayed. PHA was obtained from Difco Ltd. The same batch of PHA was used throughout these experiments. In preliminary experiments, a 1/10 dilution and 1/50 dilution added to 105 PBMNC gave a maximum and sub maximal response, respectively. A 1/10 dilution is approximately equal to 5/~g PHA/ml final concentration in the incubations. 401

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RESULTS

Effect of TxAe Receptor Stimulant U46619, TxA 2 Receptor Blocker EP045 and TxA2 Synthesis Inhibitor UK37248 on Mitogen-lnduced Lymphocyte Proliferation To characterize the role of TxA2 in mitogen-induced LP, U46619 and PHA (1/10) were added to PBMNC cultures. Inhibition of proliferation (0.1-10/~g/ml) was concentrationrelated and the IC50 was >1 10/~g/ml (N = 8). EP045 (100 nM-10/~M) did not affect LP in similar experiments and did not alter PGE2 or TxA2 synthesis (N = 6). UK37248 (100 riM-100/ZM) did not alter LP at concentrations that completely inhibited TxA2 synthesis (1/~M) but this concentration increased PGE2 synthesis in the absence of PHA in 48 hr from 38 nM+ 13.9 (SE) in control to 76.1 nM+ 26.4 (p < 0.05). These results led us to question the absence of an inhibitory effect of UK37248; particularly because the amounts of PGE2 present after 48 hr stimulation with UK37248 should be sufficient to inhibit mitogen-induced LP. We had previously demonstrated this in experiments showing that exogenous PGE2 added with PHA at time zero to PBMNC inhibited the induced LP in a concentrationdependent manner. Thus, l nM and 10 nM PGE2 inhibited LP by about 18 and 40%, respectively (N = 4), and in a current series of experiments (Fig. 1) PGE2 (50 riM) inhibited PHA-induced LP by 50% when added together with a 1/10 or 1/50 dilution of PHA. In order to resolve this apparent anomaly, we determined: (a) the effect of PGE2 added at intervals after the addition of PHA; and (b) the time course of synthesis of PGF-a and T x A 2 in PBMNC in the absence and presence of PHA and UK37248.

Time Course of Effect of PGE2 on PHA-Induced Lymphocyte Proliferation Figure 1 shows that the inhibitory effect of PGE2 (50 riM) was considerably reduced if it was added 4 hr after a 1/10 concentration of PHA rather than with PHA at time zero (P < 0.01). With a 1/50 concentration dose of PHA, inhibition of induced proliferation

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once either at the same time as the mitogen or after 4, 16, 30, 48 hr had elapsed. 3H-Thymidine uptake was measured between 48-72 hr as an assessment of proliferation. Histograms show percentage inhibition ± SE compared with control 3H-thymidine uptake. With 1/10 PHA, inhibition by PGE2 added at 4 hr was significantly less than when it was added with the mitogen (P < 0.01). With 1/50 PHA, inhibition with PGF.~ was significantly less at 16 hr than when it was added with the mitogen (P < 0.01).

The role of prostaslandins and thromboxane A 2

403

by PGE2 was markedly reduced if it was added between 4-16 hr after PHA. The inhibitory doses of PGE2 were generally more effective against lower suboptimal concentrations of PHA.

Synthesis of P G E 2 and TxA2 by PBMNC Figures 2 and 3 show that there is a lag phase in culture before substantial amounts of PGE2 or TxA2 are synthesized but between 4 and 30 hr of culture synthesis is roughly linear. At 4 hr of culture less than l0 nM PGE2 had been formed. PHA (1/10) had only a small (and statistically nonsignificant) stimulatory effect on P G ~ and TxA2 synthesis compared to controls.

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FIG. 2. 106 PBMNC were incubated for 0, 4, 16, 30 or 48 hr alone or in the presence of PHA, UK37248 (I/~M) or PHA +UK37248. Supernatant culture medium was measured for PGF~ content by RIA. At 4 hr onwards, PGE2 content, when UK37248 was present, was significantly greater than the respective controls: "P < 0.05, * ' P < 0.01 and " * ' P < 0.001 (N = 4-8). 200

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F]o. 3. Supcnlatant culture medium from experiments shown in Fig. 2 was assessed for TxA2 content by KIA. TxA2 synthesis was significantly inhibited by UK37248 when compared with the respective controls: *P < 0.05, " ' P < 0.01 and " ' * P < 0.001 (N = 4-8).

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W. Awara et al.

Effect of UK37248 on PGE2 and TxA2 Synthesis Figure 3 shows that the TxA 2 synthesis inhibitor UK37248 (1/zM) inhibited TxA~ synthesis by almost 100% at all time points in the 48 hr culture and significantly enhanced PGE2 synthesis (Fig. 2). Again, the pronounced enhancement of synthesis occurred after a lag phase of about 4 hr.

Indomethacin In two experiments where indomethacin (5#g/ml) was added together with PHA (1/5-1/50) at time zero of culture, a variable stimulation of PHA-induced lymphocyte proliferation was seen of between 16 and 44%. DISCUSSION

We have shown that the synthesis of PGE2 in control PBMNC and in the presence of PHA or UK37248 proceeds in a linear manner from 4 to about 30 hr of culture but synthesis is low before 4 hr in untreated or treated cells. Because we have also shown that the inhibition of PHA-induced proliferation by P G ~ falls off rapidly if the P G ~ is applied approximately 4 hr after PHA, it is our contention that: (1) endogenously synthesized PGE2 in control cultures cannot generally have a substantial inhibitory effect on lymphocyte proliferation because it is present in insufficient concentrations up to 4 hr. This is more likely to be true at higher concentrations of PHA. (2) Endogenous PGE2 could be effective if sufficient concentrations were attained during the initial 4 hr but even UK37248 which substantially stimulates P G ~ over a 48 hr period does not result in sufficient PGE2 being synthesized within 4 hr. (3) Indomethacin, which is widely used to identify roles for the endogenous prostaglandins in modulating mitogen, induced proliferation cannot be expect~l to have a dramatic effect on LP if endogenous prostaglandin PGE2 is unimportant in this system. Our findings clearly explain why Goodwin et aL 4 found that indomethacin potentiates suboptimal concentrations of PHA more than optimal concentrations. (4) The time course of effectiveness and synthesis of other eicosanoids that have been implicated in modulating LP must be carefully carried out. The many reports that have been published of the effects of PGE2 and TxA2 synthesis inhibitors on mitogen-induced PBMNC proliferation and utilizing the system we describe, cannot be relied upon to have given satisfactory information of the role of endogenous prostaglandins and TxA2 in the modulation of mitogen-induced LP. Acknowledgements--We appreciate gifts of UK37248, U46619 from Pfizer and the Upjohn Company, respectively. EP045 was kindly supplied by Dr R. Jones, Edinburgh University.

REFERENCES 1. 2. 3. 4. 5. 6. 7.

AKBAR, A. N. and JONF~, D. B. Immunology 51, 361-366 (1984). GOLDYt,rE, M. E. and STOSO, J. D. CRC Crit. Rev. Immun. 2, 189-223 (1981). GOODWIN,J. S., CUEPPENS,J. L. and GUALDE, N. Adv. Inflamm. Res. 7, 79-92 (1984). GOODWlN, J. S., MESSN~, R. P. and PF.AKr~,J. T. J. Clin. Invest. 62, 753-760 (1978). GORDON, D., NOURI, A. M. E. and THOMAS, R. U. Br. J. PharmacoL 74, 469-475 (1981). HmLmR, K., SMITH, C. L., JEWEI.I,, R., Aa~I"dUI~,M. J. P. and Ross, G. Gut 26, 237-240 (1985). KELLY, J. P., JOHNSON, M. C. and PARKER, C. W. J. Immun. 122, 1563-1571 (1979).