Leukotrienes are active in the rosette inhibition assay mimicking the action of ‘early pregnancy factor’

Vol. 167, No. 2, 1990 March 16, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 535-542

LEUKOTRIENES ARE ACTIVE IN TEE ROSETTEINHIBITION ASSAY HIIICKING TEE ACTION OF ‘EARLY PREGNANCY FACTOR’ Frank Clarke,

Ian Cock, Anthony Perkins and Carlos Orozco

Division

of Science and Technology, Griffith Australia

Received

January

23,

University,

NATHAN QLD 4111,

1990

Lipoxygenase metabolites have been found to be active in the rosette inhibition assay by inducing increased rosette inhibition titres. Leukotrienes B4, Cd, D4 and E4 were identified as possessing this activity. The cycloxygenase products, prostaglandins Ez, Dz and Faa had no such activity; however, prostaglandin Ez and to a lesser degree prostaglandin Dz could counteract the activity of the leukotrienes in this assay. The identified leukotrienes are the first characterised molecules known to display activity in the assay. In this respect they mimic the action of early pregnancy serum, an action ascribed to a so called ‘early pregnancy factor’. 0 1990

Academic

Press,

Inc.

In 1976 Morton et al (1) described the detection activity

in maternal serum which appeared within

of a lymphocyte modifying hours of fertilisation.

The

activity was detected by use of the rosette inhibition assay which measures the ability of antilymphocyte sera (ALS) to inhibit rosette formation between lymphocytes and heterologous red blood cells. in pregnancy sera prior

to testing

When lymphocytes are incubated

in the assay the ability

of a given ALS to

inhibit rosette formation is greatly increased resulting in the observation of an increasled rosette inhibition titre (RIT). While this activity in pregnancy sera has g’enerally been ascribed (2, 3) to the presence of a so called (early pregnancy factor’, the molecular basis for the expression of this activity, which may well be multi-factorial (4), has, until recently, remained ill defined. In 1986 (5) we found that the expression of ‘EPF’ activity in maternal serum, and the release of PAF acether from the fertilised ovum and developing

embryo which had been documented by O’Neill (6), were related induced phenomena. When PAF was injected into mice in oestrous it rapidly the expression of (EPF’ activity in the serum. Recently we have found that a combination of PAF and sera from oestrous mice applied to mouse spleen cells can mimic the ability of pregnancy sera by inducing the expression in vitro, PG, prostaglandin; PAF, platelet activating ABBREVIATIONS: LT, leukotriene; factor; NDGA, nordihydroguaiaretic acid; ‘EPF’, ‘early pregnancy factor’; RIT, rosette inhibition titre; PBS, phosphate buffered saline; HBSS, Hanks balanced salt solution; ALS, anti-lymphocyte serum; BSA, Bovine serum albumin. 0006.291x/90 535

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of an elevated

In studying

RIT(7).

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the mechanism of action

of PAF and the

oestrous mouse serum components it was found that PAF acted to stimulate the production of soluble factors which by themselves could induce elevated RIT’s The PAF- stimulated cell population if applied to fresh spleen cells. however, was rendered refractory to the action of these factors and did not display increased RIT’s unless oestrous mouse serum was added, with the latter components factors

to

production &IT’s,

was

inhibitors extractable metabolites

acting

to reverse

work. of these inhibited

the

Further

studies

soluble

factors,

by treatment

but not by cycloxygenase

refractory

state

allowing

(8) revealed that which by themselves of

the

inhibitors.

the

PAF induced

the PAF - stimulated could induce elevated

spleen cells Moreover,

with these

lipoxygenase factors were

in organic solvents suggesting that they may well be lipoxygenase of arachidonic acid. Here we report on the ability of known

arachidonic acid metabolites to mimic the effects of pregnancy serum inducing the expression of elevated RIT’s in the rosette inhibition assay.

by

MATERIALS AND METHODS nordihydroguaiaretic acid, arachidonic acid and Materials: Indomethacin, prostaglandins Ez, Dz and Fzo uere obtained from Sigma Chem Co (St. Louis, MO. , USA). Indomethacin was prepared as a 25 mM stock solution in ethanol and freshly diluted to a working solution of 0.5mM in 50% ethanol in PBS just before use. NDGA was prepared as a 4mM stock in ethanol and diluted to 0.4mM in PBS before use. Arachidonic acid was prepared and diluted in ethanol. The prosta landins were prepared as 2 mM stock solutions in ethanol and diluted in PBS/BSfi (0.1 m /ml) before use. The leukotrienes LTB4, LTC4, LTD4 and LTE4 were obtained fpram Amersham (UX). These were diluted in PBS/BSA for testing. [5,6,8,,11,12,14,15(n)-aH] LTB4 and [5,6,8,9,11,12,14,15(n)-3H]LTC4 were also products of Amersham. Cell incubations and the rosette inhibition assay: Mouse spleen cell suspensions were prepared from male mice as described previously (5 . For the standard cell incubation for the rosette inhibition assay 1 ml a 1’iquants of cells (15 x 106) were sedimented and the supernatant aspirated. The cells were resuspended in a final volume of 200~1 PBS containing the test sample(s) at the desired concentration(s). After a 30 min. incubation at 37°C the cells were sedimented and washed twice with 2 ml Hank’s balanced salt solution HBSS) , then resuspended in 1.5ml HBSS and dispensed into the ALS dilutions i or the rosette inhibition assay as described in etail elsewhere (5,7). To determine the effects of the inhibitors, indometh i?tin and NDGA were added to the cell incubations 15 min. prior to the addition of the test substance after which the incubations were continued for a further 30 min. before proceeding as above. The assay results are expressed as the rosette inhibition titres (RIT’s) defined as the highest ALS dilution in which the number of rosettes is <75% of the number formed in the controls in the absence of ALS. In keeping with past practice (5,7) the RIT has been expressed as the log 2 [reciprocal dilution of ALS x 10-31. Spleen cells incubated in PBS alone have an &IT of 12.1 l 0.4 (n = 120). Consequently the observation of an RIT > 16 was considered significantly higher than the control. Metabolism of leukotrienes: To examine the metabolism of LTB4 and LTC4 by mouse spleen cells, samples of [3H -LTB4 or [sH]-LTC4 were added to the test incubations of these substances. T1,ese incubations were terminated at various times up to 30 minutes by addition of 200~1 of ice cold, acidified methanol 536

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BIOCHEMICAL

167, No. 2, 1990

AND BIOPHYSICAL

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(methanol: acetic acid, 1OOO:l). Following centrifugation the supernatants were injected onto a Bio &ad ODS Cl8 reverse phase column (250 x 4 mm) which was developed isocratically with a solvent of 65% methanol, 0.1% acetic acid, pH5.6

(with

NHa) at

1 ml/ruin.

collecting

1 ml fractions.

Aliquants

of these

fractions were taken for scintillation counting to determine distribution of 3H -metabolites, with the column having been calibrated by running authentic f eu z otriene standards from the commercial sources listed above. RESULTS

Vhen mouse spleen the

rosette

cells

inhibition

were incubated assay,

the

with arachidonic

rosette

inhibition

acid titre

prior @IT)

to of

use in the

ALS

determined on these cells was found to be greatly increased over that determined on control cells incubated in the absence of this compound. The data in figure 1 show the dose-dependent ability of arachidonic acid to induce these elevated BIT’s with doses >lO-81 consistently being effective. If the cells were pretreated with the lipoxygenase inhibitor, NDGA, before the addition of arachidonic acid then no increase in RIT was observed (figure 1). On the other hand if cells were treated with the cycloxygenase inhibitor, indomethacin, no inhibition of the ability of arachidonic acid to induce elevated BIT’s was observed, rather the range over which arachidonic acid was effective was significantly increased with doses as little as lo-lOllI inducing increased titres (figure 1).

30 r

26 -

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22 -

18 -

14 -

+NDGA

lOJ-

$14

IO“5

,;-I3

,A-12

,;-11

,;-10

,;-9

[Arachidonic Acid] Figure

1.

,;-6

,A-7

,;-6

,A-6

,o-4

M

Dose response effects of arachidonic acid on the EIT of mouse with indicated spleen cells. Cells were incubated concentrations of arachidonic acid for 30 min. either alone or following 15 min. pretreatment with lo@ indomethacin (+INDO) or 209 nordihydro guaiaretic acid (+NDCA). Values (mean * s.d.) are from 3 independent experiments with different spleen cell preparations.

537

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Incubation of mouse spleen cells As resulted in increased RIT’s. LTB4 and the peptidoleukotrienes While there of elevated RIT’s. responses to 10-141.

the lowest

effective

AND

BIOPHYSICAL

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with individual lipoxygenase shown by the dose response

metabolites also data in figure 2,

LTC4, LTD4 and LTE4, were all were some variations in the

concentrations

were

all

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potent inducers individual dose

in the range of lo-12

a 30LTW 26-

+ pe

22-

18 -

14 -

POE2 6---------6---------d

I 10 I 10 -16

1: -15

I 10 -‘4

I 10 -I3

I 10 -12

1; -11

F’GD2 I 10-10

I 104

I 108

PGF2ci 10-7

Concentration M b CO-

18 -

10 ;

I 10 -15

10 -16

Figure

2.

I 10 -‘4

I 10 -13

I &12

,;,-11

10-10

1

I

106

104

10-7

Concentration M Effects of leukotrienes and prostaglandins on the BIT of mouse spleen cells. Cells were incubated with the indicated concentrations of each test substance alone for 30 min. before testing in the rosette inhibition assay. (a) LTB4, PGE2,PGD2 and PGFzn, (b) LTC4, LTD4and LTE4. In each case values (mean * s.d.) are from 3 - 4 independent experiments. 538

Vol.

167,

No.

As some leukocytes

2, 1990

of

BIOCHEMICAL

these (9,10)

preparations

AND

leukotrienes are their metabolism

was assessed

known

to

RESEARCH

be metabolised

by the present studies by adding

with

approximately

by

various

samples

respective test incubations and of [3H]-label in known metabolites

subsequently by means of

these

reverse phase HPLC analysis as described in Methods. there was some metabolism of LTB4 during its 30 min. cells

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spleen cell of [3H]-LTB4

during

and [3H]-LTC4 to their determining the distribution

BIOPHYSICAL

40 - 50X being

converted

mouse

As shown in figure 3a, incubation with spleen

to its

o-oxidation

products

(9). LTC4 on the other hand was rapidly metabolised when it was added to the mouse spleen cells. By 10 min. of incubation (figure 3b) it had been completely converted to LTD4 with only a little LTE4 having been derived from this latter metabolite at this time. By the end of the 30 min. incubation period (figure 3c) considerably more LTE4 (20 - 30X) had been formed, but the major metabolite was still LTD4.

60-

b

I LTC4

I LTD4

1 LTE4

50-

I

yc

LTB4

20-

E 3o 8 . s

5. 0 s

20-

IO-

O-

0

10

20

Fraction

Figure

3.

30

0

40

10

No.

Fraction

20

No.

30

Reverse phase HPLC analyses of [SE]- leukotriene metabolites following incubations of mouse spleen cells with (a) [SE]-LTB4 for 30 min. (b) [3H]-LTCd for 10 min (c) [SE]-LTC4 for 30 min. Results

are representative

of 2-4 experiments.

539

40

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I 10-13

G-11

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18

14

10 ; 10 -16

1 10 -15

Figure 4.

In

contrast

I 10 -14

to

inhibition 2a) these

the

rosette

the

absence

the

lb

lb

lb

105

potent

of

effects of these lipoxygenase metabolites in incubations of mouse spleen cells with the PGE2, PGD2 and PGF2a were without effect on the

titre. When tested over a wide range of concentrations prostaglandins had no effect on either rosette formation or

inhibition

themselves certain of the leukotrienes

titres

these

which

were

compounds.

of the prostaglandins to induce elevated

by LTD4. PGDz also displayed PGF2a was without effect over

the same as for

While

this the

they

did

not

cells

incubated

influence

the

in RIT

tested could counteract the ability &IT’s when they were added to the cell

incubation together with the leukotriene. dose dependent ability of PGE2 to inhibit

(figure

I 104

lb

Effects of prostaglandins on ability of LTD4 to induce elevated BIT. The indicated concentrations of prostaglandins Ez, Dr or F2a were added simultaneously with lo-10 M LTD4 to mouse spleen cells and incubated for 30 min before determining the RIT. Values (mean * s.d.) are from 3 independent experiments.

inducing elevated RIT’s, cycloxygenase metabolites rosette (figure

I 10-12

Figure 4 shows, for example, the the increase in BIT normally induced

ability but was far range and under the

less effective and conditions tested.

4). DISCUSSION

This study arachidonic

confirms previous acid metabolism

suggestions are active

inducing the expression of an elevated RIT. with arachidonate itself was sufficient 540

(8) that lipoxygenase products of in the rosette inhibition assay While incubation to cause an

of spleen increased

cells RIT,

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theinhibitor studies indicated that the effect was being mediated via the lipoxygenase pathway, possibly by the metabolism of the exogenously added arachidonic acid and/or by the known ability (11) of this compound to stimulate that pathway. While addition of NDGA abolished the action of arachidonic acid, addition of indomethacin extended the effective concentrati.ons range of arachidonic acid action. This latter effect could be due to increased channeling of arachidonate through the lipoxygenase pathway and/or to the inhibition of the production of counteracting cycloxygenase metabolites. The latter possibility is given credence by the observed ability of some prostaglandins to counteract the action of leukotrienes, while the former explanation is consistent with what has been frequently observed in other systems (12).

While the active lipoxygenase products generated on cell incubations with arachidonate have not been identified, it is evident from the other studies reported that well characterised leukotrienes, LTB4, C4, D4 and E4 are all capable of inducing elevated EIT’s. Whether LTC4 acts directly or via its metabolite LTD4 is difficult to discern because of its very rapid metabolism to this active metabolite. The slower rates of metabolism demonstrated from the other leukotrienes (B4, D4, E4) suggest that their actions are direct and not mediated by a metabolic product. The observed potency of these leukotrienes in this assay is consistent with the previously reported actions of these compounds as potent regulators of a variety of lymphocyte functions For example, LTB4 is known to induce proliferation of suppressor (13,14). T-cells and inhibit the generation of helper T cells over concentration ranges similar to those reported here to be effective in the rosette inhibition assay are the first identified molecules shown (14, 15). As such these leukotrienes to be active in the rosette inhibition assay where they act to induce an increase in the rosette inhibition titre. In this respect they mimic the action of early pregnancy serum. While the identified leukotrienes are not specific to pregnancy serum, the discovery of their activity in this assay used to define the ‘early pregnancy factor’ phenomenon, does provide insights into the mechanism of action of pregnancy serum and so should contribute to the resolution of the molecular basis for so called fearly pregnancy factor’ activity expression.

ACKNOWLEDGIENTS This work was supported of Australia.

by the National

541

Health

and Medical

Research Council

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REFERENCES 1.

Morton, II., 413- 419.

Hegh, V. and Clunie,

2.

Morton, (1977)

3.

Morton, II., Rolfe, Biol. 23, 73-92.

4.

Clarke, F.E. and Wilson, S. (1985) Ellendorf & E. Koch, eds) pp. 165-177.

5.

Orozco, C., 549- 555.

6.

O’Neill,

7.

Orozco, C., Cock, I., Fert. 88, in press.

8.

Clarke, F.P., Orozco, C., Perkins, Fert. 88, in press.

9.

Shak, S. and Goldstein,

10.

Nagaoka, I. 282- 287.

11.

Kok, P.T.R., Hamelink, I.L., and Bruynzed, P.L.B. (1988)

12.

Kuehl, l-5.

13.

Payan, D-G., Hissirian- Bastian, Acad. Sci. USA 81, 3501-3505.

14.

Goodwin, J.S.

15.

Rola-Pleszczynski, P., Borgeat, P. Biophys. Res. Commun. 106, 1531-1537.

II., Rolfe, B., Clunie, Lancet i, 394- 397.

F.A.,

G.J.A.,

B. and Cavanagh,

Perkins,

C. (1985)

G-J .A. (1976)

T. and Clarke, J. Reprod. Perkins,

I.M.

and Yamashita,

Dougherty,

Fert.

Anderson, A.C.

F.M.

(1986)

542

B. 193,

Topics

J.

Devel.

J.

Reprod.

Fert.

(F. 78,

73, 567-577. F.P.

(1990)

J. Reprod.

A.V. and Cock, I.

(1990)

J. Reprod.

J. Biol.

Chem. 259, 10181-10187. Res. Commun. 147,

A.M., Verhagen, J., Koenderman, L. Biophys. Res. Commun. 153, 676-682.

A. and Goetzl,

T. (1988)

Sot.

and Morrison,

Curr.

T. (1987) Biochem Biophys. Kijne, Biochim.

Il.

In Early Pregnancy Factors Perinatology Press. New York.

H.W. and Ham, E.A. (1984)

and Behrens,

M.J.

(1987)

A.V. and Clarke,

(1984)

Proc.

Biochem. E.J.

(1984)

Ann. N.Y. Acad. Sci. and Sirois,

Pharmaol.

P.

Proc.

33, Natl.

524, 201-207.

(1982)

Biochem.