The effects of human seminal plasma and PGE2 on mitogen induced proliferation and cytokine production of human splenic lymphocytes and peripheral blood mononuclear cells

Journal of Reproductive I 30 (1996) 97- 114

engshun

iao,

Department of SW,PTy, University of Edinburgh dical School, Teviot Place, Edinburgh, EH8 9AG, UK Received 20 November 1995; revised 11 January 1996; accepted 24 January 1996

Abstract The effects of human seminal plasma (HSP) and prostaglandin E, (PGE,) on the proliferative responses of human splenic lymphocytes and peripheral blood mononuclear cells (PBMCs) to phytohaemagglutinin (PHA), anti-CD3 mAb and anti-CD3 plus anti-CD28 mAb have been studied. Thl and Th2 cytokines were also measured in the supernatants of selected cultures. Both HSP and PGE, reproducibly inhibit the proliferative response to PHA and anti-CD3 mAb in a dose dependent manner. These effects were observed with both fresh and frozen human PBMCs and splenic lymphocytes. HSP and PGEz however were less effective in inhibiting the co-stimulatory response induced by anti-CD3 plus anti-CD28 mAb. In addition, the HSP and PGE2 treatment used inhibited the production of the Thl cytokines IL-2 and IFNg but had a differential modulatory effect on Th2 cytokine production, namely enhancing the production of IL-6 whilst simultaneously impairing the synthesis of IL-4 and IL-IO. Keywords: Human seminal plasma; Prostaglandin

* Corresponding

E,; Cytokine production

author.

0165-0378/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SOI 65-0378(96)00957-6

98

D. Mao et al. 1 Journal of Reproductive Immunology 30 (1996) 97-114

1. Introduction uman seminal plasma1 (HSP) and fractions derived therefrom have been n to impair the generation and activity of various cells of the immune system in vitro, including T and B cells, macrophages, polymorphonuclear leukocytes, natural killer (NK) cells and lymphokine-activated killer (LAK) cells (reviewed by James and Hargreave, 1984; Alexander and Anderson, 1987; Hargreave et al., 1993; James and Skibinski, 1995). Among the active components of HSP, prostaglandins of the E series are the best characterised and probably the most important immunosuppressive substances (Tarter et al., 1986; Vallely et al., 1988; Quayle et al., 1989; Skibinski et al., 1992; Skibinski et al., 1993). PGEs are present in HSP in uniquely high concentrations compared with any other body fluid (Templeton et al., 1978). Among PGEs, PGE2 is the most potent inhibitor, followed closely by PGEl , while the potency of 19-OH PGE is almost 100 times lower, revealed by their ID,,s in PHA-induced lymphocyte proliferation and N cytotoxic assays (Skibinski et al., 1992). PGE2 can inhibit T cell proliferation, IL-2 production and IL-2 receptor expression (Vercammen and Ceuppens, 1987), impair B cell activity and the production of IgM (Phipps et al., 1989; Roper et al., 1994), suppress the production of IL-l by macrophages (Brandwein, 1986) and NK activity (Goto et al., 1983; Skibinski et al., 1993). Overall these studies suggest that PGEZ acts as a general immunosuppressive agent. However, other studies have revealed immunostimulatory effects of PGE*, thus PGE2 has been found to stimulate the IL-4 mediated class switch of B cells and enhance the IL-4 induced production of IgGl by mouse B cells (Roper et al., 1990). In addition, it has been shown that PGE2 inhibited the production of Thl cytokines (IFN-g and IL-2) but had no effect on production of Th2 cytokines (IL-4 and IL-5) (Betz and Fox, 1991; Novak and Rothenberg, 1990). More recent reports indicate that the modulatory effect of PGE2 on Th2 cytokine production depends on the T cell population studied and the culture and stimulation conditions applied (Watanabe et al., 1994; Van der Pouw Kraan et al., 1995; Parker et al., 1995; Hilkens et al., 1995). In the present communication we have addressed several questions, namely: (1) Do HSP and PGE, affect the response of human lymphoid tissues other than human peripheral blood mononuclear cells (P Cs)? (2) Could similar effects be observed with more specific mitogens? (3) Are these effects mediated in part by altering co-stimulatory molecule activity? And finally, and most importantly, (4) Is there any evidence for a discriminatory effect of HSP and PGE2 on Thl and Th2 responses?

il. Miao et al. / Journal of'

o do this we investigated t tive response of splenic lymphocytes and ), monoclonaa a mAb mixture. supernatants of selected cultures. The results confirm that response of specific (anti stimuli in a dose de however, less effective at inhibiting the enhan observed in the anti-C inhibiting the product had a variable effect on the relea illustrate the complex effects th ulatory substances such as PGE2 and HSP can have on cytokine responses.

2.1. Source and processing of lywmphoidtissues 2. I. I. Human peripheral blood ~~o~o~~c~earcells (PB Heparinized venous blood was obtained from healt were isolated from the blood on Ficoll-Hypaque density gradients (density 1.077). After three washings in phosphate buffered s concentration was adjusted to 1 x 106/ml in complete RL, Paisley, UK) supplemented wi penicillin and streptomycin/ml. The media also contained 10% (v/v) foetal b normal human serum while in experiments employing (NHS) (Edinburgh and South East Service) was employed to avoid pos suppression (Vallely and Rees, 198 the cells were suspended at 1 x IO7 cells/ml in 10% dimethyJ sulphoxide (DMSO) in F 2.1.2. Human splenic lymphocytes Human spleens were obtained from the Renal Transplant Unit at the Western General ospital, Edinburgh. They were retrieved fro gan donors and usually reached the laboratory within two ho this time the tissue was stored in sterile sahne at 4°C. The spleen tissues were homogenized on arrival using a loose-fitting glass homogenizer. man splenic lymphocytes were isolated from the splenocytes on Ficoll

100

D. Mao et al. 1 Journal of ReproductiveImmunology 30 (1996) 97- 114

paque gradients. After three washings in PBS, the cell concentration was Cs for dium as described for P adjusted to 1 x 106/ml in the S with 10% DMSO for cryopreservaexperiments and to 1 x 107/ml in tion. 2.2. Reagents Phytohaemagglutinin (PHA), goat anti-mouse IgG, bovine serum albumin (BSA) and prostagiandin Ez (PGE,) were purchased from the Sigma Chemical Co. (Poole, Dorset, UK). The anti-CD3 mAb (mouse monoclonal antibody, clone UCTHl, IgG) and anti-CD28 mAb (mouse monoclonal antibody, IgG,) were obtained from the Scottish National Blood Transfusion .Service Reagents Laboratory, Law Hospital (Carluke, Lanarkshire) and Becton Dickinson (San Jose, CA), respectively. The PGE2 was stored at - 20°C as a 2 mg/ml stock solution in ethanol. 2.3. Human seminal plasma HSP was obtained from normal donors and supplied by the MRC Reproductive Biology Unit, Edinburgh. The spermatozoa were removed by centrifugation (1000 x g, 20 min) and pools of 30 samples were made. These pools were subsequently dispensed into 1 ml aliquots which were stored at - 20°C until used. 2.4. Measurement qf PGE, by radioimmunassay The method described by Kelly et al. (1991a) was used. Radioimmunoassay was performed on PGE protected as the methyloxime. Using this technique the PGEz concentration in the pooled HSP used in the present study was found to be 53.3 pg/ml or 1.5 x 10m4 M. 2.5. Preparation of solid phase anti-CD3 mAb plates

The wells of 96-well microtitre plates (Costar, Cambridge, UK) were coated with 50 ~1 of a 20 pg/ml solution of the anti-mouse IgG in 0.05 M Tris, pH 9.4, at 4°C overnight. After three washes with PBS the wells were blocked with 100 /II of 1% (w/v) BSA in PBS for 1 h at 37°C. Following a further three washes with PBS, 50 ,ul of O.l- 10 pg/ml anti-CD3 mAb in 1% BSA was added to the plates which were incubated for 2 h at 37°C. Finally, they were washed three more times with PBS and either used immediately or stored at 4°C for up to a week. The wells of six-well tissue culture plates were coated using the same protocol with 200 times larger volume of reagents.

2.6. proliferation msays

0th fresh and frozen red in complete (Costar, Cambridge, UK) in the absence or presence of stimuli which included A (0.25-10 pg/ml), j4g/ml) previously coa on plates and antipg/ml). The effects of the proliferation re culture. All cultures incubated at lOOoh hours before termi j&/well of [” on termination we cell harvester (Skatron, assessed by determining tritiate Ca liquid scintilla 2.7. Cytokine assays In order to obtain sufficient culture su Cs and splenic lymphocytes were also c medium in six-well culture plates (Costar, Cambridge, Engla ml of medium/well were cultured in the absence or as described in the proliferation assa supernatants were harvested by cent l-ml aliquots were stored at assayed for the presence of commercially availa Systems Inc., Abingdon, 1 assays were perfo performed in parallel on these samples.

and PGE, on PHA induced p~ollfe~atio~of human splenic lymphocytes and

3.1. Inhibitory eflects of

The overall results of t se studies are summarised in Table 1 w of a typical experiment are presented in Fig. 1.

+ f + + f f

Peripheral blood lymphocytes 3 87.2 0.25 2 56.6 1.0 2 39.1 2.5 3 45.1 5.0 3 50.8 10.0 55.9 Mean 6.4 7.8 7.8 6.4 6.4 3.6

6.4 4.9 4.2 4.9 4.5 2.6 70.4 33.3 13.3 20.5 26.3 33.2

43.2 29.2 30.0 36.5 50.9 39.0

6.4 4.5 3.9 4.5 4.5 2.4

f 6.4 + 7.8 + 7.8 h 6.4 &- 6.4 f 3.6

f f * + + + 44.8 25.8 8.4 15.4 18.3 21.6

27.4 18.5 15.6 24.6 19.0 22.1 + k _t + 2, _t

7.8 11.1 7.8 7.8 7.8 4.3

+ 6.4 _t 4.5 k 3.9 + 4.5 + 4.5 -1_ 2.4 1 1 1 1 1

2 3 3 2 2

+ + f + + +

5.2 4.2 4.2 5.2 5.2 2.2

99.9 94.3 78.3 48.8 31.9 70.7 & 3.3

94.3 83.8 71.0 56.8 75.3 76.2

1%

+ + f + + f

5.2 4.2 4.2 5.2 5.2 2.2 77.7 58.2 25.3 8.6 8.9 35.7 & 3.3

58.9 39.0 38.7 38.7 38.2 42.7

0.1%

3.3

+ 5.2 + 5.2 & 4.2 f 5.2 f 5.2 -I_ 2.2 29.5 17.0 0.3 0 0 9.3 f

34.8 21.5 13.4 17.4 17.4 21.5

0.01%

2 x 10’ frozen human splenic lymphocytes or fresh human PBMC were cultured with various concentrations of PHA in the presence of various concentrations of PGE2 or HSP. Data are expressed as percentage inhibition (mean + SE.) of the control response in the absence of PGE, or HSP. The values presented are for day 3. Representative counts incorporated are p-ortrayed in Fig. 1 and Tables 3 and 4. Note ‘n’ refers to the number of occasions on which experiments were performed under the condkions indicated.

+ * + * f f

84.4 79.7 89.3 85.8 94.6 89.1

10-8M

n

10-6M

n

10-5M

HSP

of human splenic lymphocytes and PBMC

PGE

Splenic lymphocytes 3 0.25 5 1.0 9 2.5 5 5.0 7 10.0 Mean

PHA (it g/ml)

Table 1 The inhibition effect (%) of HSP and PGEz on the PHA induced proliferation

PGE2 concentration (M)

-

sp1e!fznhiay5 c/day 5

Fig. 1. The effect of HSP and PGE2 on the PHA-induced proliferation of human splenic lymphocytes and PBMC. 2 x 105 frozen human splenic lymphocytes or fresh PBMC were cultured with 1 pg/ml PHA in the presence of various concentrations of PGEz or HSP. Data are expressed as mean counts/min of quadruplicate cultures and percentage inhibition referred to the control response in the absence of PGEz or HSP. HSP experiments used 10% HS supplemented medium, while the PGE2 experiments were performed in media containing 10% FBS. In all experiments the S.E.s were always lower than 10% and have been omitted for clarity.

104

D. Miao et al. / Journal of Reproductive Immunology 30 (1996) 97-114

Although the degree of proliferation varied from experiment to experiment, being greatest in fresh cell cultures in F S supplemented m and HSP consistently inhibited the prolife tive response in spleen. The inhibition achieved was directly related to the amou and HSP added to the cultures. There was evidence however that the inhibition achieved in PBL cultures was less with higher doses of mitogen, that is in cultures exhibiting the greatest proliferation. A number of additional points are worthy of note. In the first place the optimum doses of mitogen varied from tissue to t ue, being I- 1.5 PgElml for spleen and 5- 10 p g/ml for peripheral blood. rthemore, the use of frozen spleen cells in most of the studies reported here was necessitated by the difficulty in obtaining regular supplies of fresh human spleen. However, it should be stressed that while comparative studies reveal that frozen and spleen exhibit a poorer proliferative reponse than fresh cells, the pattern of inhibition with PGE2 and HSP was similar (data not included). 3.2. Inhibitory eflects of HSP and PGE, on anti-CD3 mAb and anti-CD3 plus anti-CD28 mAb induced proliferation of human splenic lymphocytes and PBMCs

The effects of HSP and PGE2 on the more specific mitogenic stimulus induced by anti-CD3 are illustrated in Table 2 and Figs. 2 and 3. They were both found to effectively inhibit the proliferative response of spleen and peripheral blood lymphocytes to a range of doses of anti-CD3 antibody. Once again the inhibition observed was directly related to the amounts of PGE, and HSP added to the cultures. As expected, the addition of anti-CD28 to the anti-CD3 system generally enhanced proliferation (Figs. 2 and 3). Of particular interest however was the observation that the inhibition in the co-stimulatory model was always less than that noted with anti-CD3 alone. This was particularly noticeable in experiments involving PGE2 (Fig. 3; Table 2). It should also be noted that anti-CD28 alone failed to induce proliferation in either spleen or peripheral hocytes. 3.3. The eflects of HSP and PGEz on mitogen induced cytokine production in human spleen and peripheral blood lymphocytes

These studies on selected culture supernatants revealed that HSP exerted a differential effect on cytokine production in mitogen stimulated human lymphocyte cultures. Generally speaking they inhibited the synthesis and secretion of the Thl cytokines IL-2 and IFNg and the Th2 cytokines IL-4 and IL-IO. In marked contrast, on all but one occasion they

Time (days)

88.1 93.9 4.5 13.7

1

44.4 f

2 1 1 1

100 100 90.0 92.9 87.7 81.9 70.8

+ + + + + 2 f

1O-5 M

3 2 5 5 3 2 2

n

PGEz

10

6.7 7.4 5.2 4.7 6.7 7.4 3.0

+ 6.7 + 7.4 &- 5.2 * 4.7 + 6.7 + 7.4 i: 3.0

13.5

60.6 47.5 0

24.0 & 10

85.3 88.2 63.9 31.9 66.9 13.2 36.0

lO--‘j M

+ 6.7 &- 7.4 + 5.2 * 4.7 + 6.7 9 7.4 1_ 3.0

11.5

13.4 9.8 0

0 + 10

49.5 55.3 30.5 15.1 38.9 3.5 4.8

lo-* M

1

1 1 1

4 2 I 2

83.0

95.6 99.0 91.2

32.3 + 11

7.6 15 6.2 8.8 7.6 15 14

2

+ f + + f + +

100 100 93.2 96.6 96.0 97.8 60.5

1%

2 1

n

HSP

+ + f + & + _+

7.6 15 6.2 8.8 7.6 15 14

48.8

91.1 97.7 55.8

19.0 t_ 11

89.9 79.5 66.4 79.2 80.3 78.0 25.0

0.1%

t_ + f & k + k

7.6 15 6.3 8.8 7.6 15 14

13.4

56.4 83.8 10.3

3.7 + 11

71.4 19.6 50.0 38.6 49.3 23.9 12.8

O.Ol’%

of human

*Anti CD3 (1.0 p&/ml) plus anti CD28 (0.1 pg/ml) 2 x IO5 frozen human splenic lymphocytes or fresh huma C were cultured with various concentrations of anti-CD3 mAb previously coated on plates in presence or absence of 0.1 pg/ml antimAb in the presence SP. Pooled data from a number of experiments are presented as percentage f S. of various concentrations of PGE2 o P. Further details of the counts incorporated and cytokine produced are to be fou responses in the absence of PGEz or 3 and Tables 3,4. When anti C 3 plus CD28 studies were performed parallel studies with anti C 3 alone were always incl direct comparison.

Peripheral blood lymphocytes Anti CD3 (1.0) 3 5 Anti CD3 + 3 CD28 5

Splenic lymphocytes Anti CD3 (0.1) 3 5 Anti CD3 (1.0) 3 5 Anti CD3 (10) 3 5 Anti CD3 + 3 CD28* 5

Mitogen @g/ml)

Table 2 The inhibition effect (%) of HSP and PGEz on the anti-CD3 mAb and anti-CD3 plus anti-CD28 mAb induced proliferation splenic lymphocytes and PBMC

D. Miuo et al. ] Journal of Reproductive lmmunoiogy 30 (I 996) 97- $14

day 5

day 3 250 225 ‘Loo 175 150

125 100

75 50 25 0 loogo-

800 706050-I 40, 30-

4

HSP concentration (%) -II==

CD3/spleen

m*nm CD3+28/spleen

lu*+8

CD3/PBMC

I-+-

CD3+28/PBMC

Fig. 2. The effect of HSP on anti-CD3 and anti-CD3 plus anti-CD28 induced proliferation of human splenic lymphocytes and PBMC. 2 x IO5 fresh human splenic lymphocytes or fresh PBMC were cultured with either 1 pgjrnl anti-CD3 monoclonal antibody or 1 pug/ml anti-CD3 plus 0.1 p&ml of anti-CD28 monoclonal antibody in the presence of varying concentrations of HSP. Data are expressed as mean counts incorporated into quadruplicate cultures and as a percentage inhibition of the value observed in control cultures in the absence of HSP. S.E.s of the mean were always less than 10% and have been omitted for clarity.

D. Miao et al. / humal

of

100

90 80 70 60 50 40 30 20 10 0 0-7

10-6

IO-5

2 co CD3/spleen

-"

CD3+28/spleen

... 0'.CD3h'BMC Fig. 3. The effect of PGE, on anti-CD3 and anti-CD3 plus anti-CD28 induced proliferation of human splenic lymphocytes and PBMC. 2 x IO5 fresh human splenic lymphocytes or fresh PBMC were cuhured with either 1 peg/ml anti-CD3 monoclonal antibody or 1 /lg/ml anti CD3 plus 0.11 pug/ml of anti-CD28 monocional antibody in the presence of varying concentr.ations of PGE2. Data are expressed as mean counts incorporated into quadruplicate culture and as a percentage inhibition of the value observed in control cultures in the absence of PGE2. S.E.s of the mean were always less than 10% and have been omitted for clarity.

108

D. Miao et al. ] Journal of Reproductive Immunology 30 (1996) 97-114

enhanced IL-6 production in mitogen stimulated human splenic lymphocyte and peripheral blood lymphocyte cultures (Table 3 and Table 4). A number of other additional points emerged from these studies and are worthy of comment. In general, much higher levels of IFN-7, IL-2 and IL-4 were observed in the spleen cultures compared with PBMCs. The latter however produced much higher levels of IL-6. Furthermore, in almost all cases greater concentrations of cytokines were observed in cells cultured in media containing FBS (Table 4) compared with NHS (Table 3). This is most apparent with respect to peripheral blood cells (Table 3). Once again IL-6 proved to be the exception with the highest concentrations being detected in PBMCs cultured in RPM1 1640 medium containing human serum. Finally it should also be noted that while anti-CD28 alone at the doses used had a negligible effect on both lymphocyte proliferation and cytokine secretion (data not included), its addition to anti C cultures resulted in a marked increase in the secretion of the Thl cytokines IL-2 (27-107 times) and IFN-7 (3-20 times).

4. Discussion In the present study we have shown that HSP impairs the proliferative response of both human spleen and peripheral blood lymphocytes to a mitogenic stimulus other than plant lectins, namely antibody against the T cell receptor. This was achieved with O.l-1.0% v/v amounts of HSP which would give final concentrations of PGEz in culture of 1.5 x 10 - ’ to 1.5 x lo- 5 M. As anticipated, similar effects were also observed with approximately equivalent amounts of purified PGE2, the major immunosuppressive component in human seminal plasma, thus confirming previously published observations with this compound (Vercammen and Ceuppens, 1987; Watanabe et al., 1994). Our results also indicate that the inhibitory effects of HSP and PGE%can be partially overcome by the addition of antibodies such as anti-CD28 which deliver co-stimulatory signals. Nevertheless, as previously observed, marked inhibition still occurs (Minakuchi et al., 1990). Studies from our own laboratory and elsewhere have indicated that PGE, impairs mitogen-induced lymphoproliferative responses in vitro by inhibiting the production of IL-2 and IL2R at both the messenger RNA and protein product level (Rappaport and Dodge, 1982; Chouaib et al., 1985; Quayle et al., 1989; Anastassiou et al., 1992; Vercammen and Ceuppens, 1987). While the present results provide further support for this contention and demonstrate the effects of HSP itself on IL-2 production they conflict with previous observations and hypotheses that PGE2 modulates immune responses by preferentially suppressing the production of Thl cytokines

99 969 9196

0

I 0. I

(2.5)

(I.O)h

PHA

CD3

CD3 (I.O)b

CD3 (1.0)’

+CD28

I

2a

2b

3a

3b

Blood

Spleen

Spleen

Blood

Blood

- 52.8 -8.6

71977 21686 54 030 79 153 37 928 72 433 16 251

0

I

0.1

0

I 0.1

0

CD3 (l.O)b

+CD28

0

- 10.3

- 33.0

-65.5

t- 4.8

- 33.7

- 97.0

-99.4

0 0

0

0

0 0

0

0

0

53

27

I26

IO

0

57

0

0

0

-41.3

-66.9

-78.5

-93.4

-100 - IO0

46

-49.7 I5

3

44 -96.7

0

75x3 0

4285

12 927

26

8

121

0

0

I

532

35

1058

pgml

( -) or enhancement ( + b.

-

- 57.9

- 78.6

- 82.5

IO0

- 100

- 100

+4.5

-93.2

‘%I

25 188

25 306

14 130

25 563

27 794

15 118

4430

7423

2702

4563

8299

2111

34231

35 357

32 059

487

642

487

p&ml

IL-6

0

0

0

0

I)

53 0

10

95

41

3

187

22

28

31

0

0

0

pg:ml

IL-10

-44.2

-89.5

-78.1

-98.4

- 29.0

-9.7

%

referred to cytokine production in cultures in the

mAb in the presence of

+78.3

+ 19. I

f69.1

+ 83.8

+63.9

+ 171.1

$116.1

+293.1

+6.8

+ 10.3

0

+31.8

‘%I

-,-_

anti-CD.1 mAb previously coated on plates with or without anti-CD28

0

-91.2

were cultured with PHA,

44416

0

- 56.4

1842 0

0

-95.6

0

44 274

2677

1693

2555

1131

-25.3

- 62.5

2540

- 18.4

- 100

‘%I

IL-4

production was measured in 3-day cultures supernatants. The percentage inhibition

“Refers to antibody against CD antigen indicated.

“Percentage of inhibition

absence of HSP.

various concentrations of HSP. Cytokine

3 x 100 fresh human splenic lymphocytes or PBMC

(0.1)’

I 0.1

IS271 29 683

-25.3

8942 30 084

I 0.1

(O.l)b

16 76

- 78.3

40 152

0

200

- 25.8

9156

0

0.1

245

2678

I

- 78.7

I2 315

0

(1.0)

PHA

I

Spleen

p&ml

‘li,

pg/ml

cotmtsimin

‘%I”

IL-2

secretion in human splenic lymphocytes and PBMC IFN-;

IL-6 and IL-10

‘HTdR

HSP (XI)

Mitogen (j~g/ml)

Exp.

IL-4.

Tissue

IL-2,

IFN-;.

Table 3 The eflect of HSP on mitogen-induced

CD3 (1.O)b

CD3 (l.O)b iCD28 (O.l)b

2a

2b

3a

3b

Spleen

Spleen

Blood

Blood

5 8 0 5 8 0 5 8 0 5 8 0 5 8

0

IO-”

81 316 50 795 77 846 51 827 21 268 36 866 86 751 8089 66931 93 522 62 072 88 546 14 4358 17 504 16 2771 14 6933 14 0287 15 6505 -4.3 0

-88.1 0

-34.5 -5.5

-93.2 -23.5

- 59.5 -29.1

-38.7 -4.4

21 778 34 20 342

7809 3653 6957 I003 148 303 2232 719 2656 46 248 7079 33 391 6585 0

-99.8 -6.6

-100 -26.2

-84.7 -27.8

-67.8 +19.0

-85.2 -69.5

- 53.2 - 10.9

4741 2150 3674 274 289 205 406 16 87 11 135 1241 4894 125 0 52 5849 125 1832

pg!ml

%

pg/ml

counts/min

‘V

IL-2

IFN-;

‘HTdR

- 97.9 -68.7

-100 - 58.4

- 88.9 - 56. I

-100 -100

+17.1 - 17.1

- 54.7 -22.5

%I 312 268 332 59 33 8 76 0 34 126 65 147 5 0 0 25 0 I5

p&ml

IL-4

and PBMC

-100 -40.0

-100 -100

-48.4 + 16.7

-100 -55.3

-44.1 -69.5

- 14.1 +6.4

‘%I

2318 5412 3042 8535 14 007 7125 1587 4851 1668 2097 5464 2437 4164 11777 6209 3787 12 663 6623

p&ml

IL-6 -

+ 234.4 + 74.9

+ 182.8 +49.1

+ 183.3 +20.5

+5.1

+ 205.7

+64.1 - 16.5

+ 113.5 +31.2

%

10 0 2 15 0 IO 232 0 75 166 5 99 102 0 19 114 2 49

pdml

IL-10

-

-98.2 - 57.0

-100 -81.5

-97.0 -40.4

-100 -67.7

-100 -33.3

-100 - 80.0

“%I

3 x IO” fresh human splenic lymphocytes or PBMC were cultured with PHA. anti-CD3 mAb previously coated on plates with or without anti-CD28 mAb in the presence of various concentrations of PGE,. Cytokine production was measured in 3-day cultures supernatants. The percentage inhibition or enhancement referred to cytokine production in cultures in the absence of PGE,. “Percentage inhibition ( - ) or enhancement ( + ). bRefers to antibody against CD antigen indicated.

CD3 (1.0)’ +CD28 (0.1)’

CD3 ( l.Qh

PHA ( 1.0)

1

Blood

5 8

0

PHA ( 1.0)

1

Spleen

PGE, M

IFN-7, IL-2, IL-4, IL-6 and IL10 secretion in human splenic lymphocytes

Mitogen (/(g/ml)

Exp.

Tissue

Table 4 The effect of PGE, on mitogen-induced

(Novak and Phipps et al., 1991). Our observations that bot the production of Thl cytokines whilst si ing the production of Th2 cytokine situation. While this discrepancy previous studies indicating selectivi should be noted that the complexit reported in other systems. For exa inhibit IL-6 production in LPS stimulated monocyte cultures whilst simultaneously enhancing the release of I or not affecting the production of others have claimed that the net effect of critically depends on t populations studied ( the effects of PGEz (an producing capacity of th Thl and Th2 cytokine of the target cells is lo support the contention that the Thl/Th2 model as first described is probably oversimplified, distinct Thl and Th2 cytokine producing cells representreduction and secretion ( ing the ends of a spectrum of cytokine 1995). The differences in cytokine response noted in the various culture systems -6) higher are also worthy of comment. With one exception (namely cytokine responses were always noted in spleen compared wi peripheral blood lymphocyte cultures. Furthermore, with both tissues greater levels of cytokine were produced in cultures supplemented with F S, with IL-6 once e present time we can only speculate on the more being the exception. ile the tissue effects may be attributed to basis of these differences. differences in cellular composition and activational status, the failure to Cs cultures in detect significant levels of cytokines other than IL-6 in NHS may be due in part to the binding (and hence ng, of certain cytokines) by soluble receptors, naturally occurring anticytokine antibodies or other interfering substances. However it is apparent from the present studies that there is no obvious relationship between the concentration of any individual cytokine in tissue culture supernatant as detected by immunoassay and proliferative response. TIIe present studies clearly indicate that the effects of stimulated spleen and peripheral blood lymphocytes purified PGE2, thus providing further evidence that prostaglandins of th series are responsible for many of the immunosuppressive effects of human seminal plasma (Tarter et al., 1986; Vallely et al., 1988; Quayle et al., 1989;

112

D. Mao et al. } Journal of Reproductive Immunology 30 (1996) 97- 114

components other than prostaglandins should not be overlooked in view of observations from our own laboratory of residual immunosuppressive activity in prostaglandin stripped HSP (Quayle et al., 1989) much of which can be attributed to prostasomes (Kelly et al., 1991b). It obviously will be of interest to determine the effects of these extracellular organelles on cytokine production. The relevance of these observations to infection of the genital tract continues to be controversial. However it is interesting to note that in addition to inhibiting the synthesis of key Thl cytokines such as IL-2 and IFNg these studies show that HSP and PGEz exert diametrically opposed effects on certain Th2 cytokines which might be relevant to HIV infection, namely enhanced IL-6 production and decreased IL-10 synthesis, changes which would favour HIV replication (Poli et al., 1990; Weissman et al., 1994). Furthermore, should HSP inhibit IL-10 production, as has recently been reported for PGEz on LPS stimulatory macrophages, its possible relevance to sexually transmitted diseases would be even greater. Of additional interest are the observations that both HSP and PGE2 may be less effective at inhibiting the co-stimulatory response induced by anti CD3 plus anti CD28. This would seem to imply that the immunosuppressive effects of such compounds may be less important if seminal, vaginal or cervical antigen presenting cells express the natural ligands for CD28, namely B7-1 or B7-2. Investigations into the expression of such molecules by reproductive tract tissue are clearly warranted. Skibinski, 1992). However the pbossiblecontributory effects of

Acknowledgements

The authors wish to thank Dr R. Kelly for performing PGE2 immunoassays and for providing the pooled seminal plasma. Dr D. Miao gratefully acknowledges the receipt of a Sino-British Fellowship Trust Award from the Royal Society.

References

Anastassiou,E.D., Paliogianni, F., Balow, J.F., Yamada, H. and Boumpas, D.T. (1992) Prostaglandin E, and other cyclic AMP-elevating agents modulate IL-2 and 11-2 R gene expression of multiple levels. J. Immunol. 148, 2845-2852. Alexander, N.J. and Anderson, D.J. (1987) Immunology of semen. Fertil. Steril. 47, 192-205. Betz, M., Fox, B.S. (1991) Prostaglandin E2 inhibits production of Thl lymphokines but not of Th2 lymphokines. J. Immunol. 146, 108-l 13.

Brandwein, S.R. (1986) Regulation of interleukin 1 production by ouse eritoneal macrophages: effects of arachidonic acid metabolites, cyclic nucleotides and interferons. J. Biol. Chem. 264, 8624-8632. Chouaib, S., Welte, K.. Mertelsmann. R. an rostagla~(~i~ E2 acts on two distinct pathways of T lymphocyte activation: inhibition of interleukin-2 production and down regulation of transferrin receptor expression. J. Immunol. 135, 1172- 1179. Gold, K., Weyand, C.M. and Goronzy, J.J. (!994) Modulation of helper T cell function by prostaglandins. Arthritis Rheumatism 37, 925-933. Goto, T., Herberman, R.B., Maluish, A. and Strong, D.M. (1983) Cyclic A P as a mediator of prostaglandin E-induced suppression of human natural killer cell activity. J. Immunol. 130, 1350. Hargreave, T.B., James, K., Kelly, R.W., Skibinski, 6. and Szymaniec, S. (19 suppressive factors in the male reproductive tract. In: Local Immunity in Tract Tissues (Griffin, P.R.D. and Johnson. P. eds.). pp. 161- 175. Oxford University Press. Oxford. Hilkens, C.M.U.. Vermeulen, H.. Neerven. R.J.J.. Snijdewint, F.G.M.. Wierenga. E.A. and Kapsenberg, M.L. (1995) Differential modulation of T helper type 1 (Thl ) and T helper type 2 (Th2) cytokine secretion by prostagla~di~ E2 critically depends on interleukin-2. Eur. J. Immunol. 25, 59-63. James, K. and Hargreave, T.B. (1984) Immunosuppression by seminai plasma and its possible clinical significance. Immunol. Today 5, 357-363. James, K. and Skibinski, G. (1995) Immunosuppressive factors in human seminal plasma: their effects, characterization and possible mode of action. In: Immunology of Human Reproduction (Kurpisz. M. and Femandez, N.. eds.), pp. 267-283. BIOS Scienti Publishers Ltd., Oxford. Kelly, R.W., Quayle, A.J.. Wallace, E.M., Wu, F.C.W., Hargreave, T.B. and James. K. (1991a) Immunosuppression by seminal plasma from fertile and infertile men: inhibition of natural killer cell function correlates with seminal PG concentration. Prostagland.. Leukotr. Essential Fatty Acids 42, 257-260. Kelly, R-W., Holland, P., Skibinski. G., Harrison. C.. McMillan. L.. Hargreave. T.B. and James, K. (199lb) Extracellular organelles (prostasomes) are immunosuppressive components of human semen. Clin. Exp. Immunol. 86, 550-556. Kelso, A. (1995) Thl and Th2 subsets: paradigms lost? Immunol. Today 16, 374-379. Minakuchi, R., Wacholtz, M.C., Davis, L.S. and Lipsky. P.E. (1990) Delineation of the mechanism of inhibition of human T cell activation by PGE,. 9. Immunol. 145. 26162625. Novak, T.J. and Rothenberg. E.V. (1990) CAMP inhibits induction of interleukin 2 but not of interleukin 4 in T cells. Proc. Natl. Acad. Sci. USA 87, 9353-9357. Parker, C-W., Huber, M.G. and Godt. SM. (1995) Modulation of IL.-4 production in murine spleen cells by prostaglandins. Cell. Immunol. 160, 278-285. Phipps, R.P. Lee, D., Schad, V. and Warner. G.L. (1989) E-series prostaglandins are potent growth inhibitors for some B lymphomas. Eur. J. Immunol. 19, 995-1001. Phipps, R.P., Stein, S.H. and Roper, R.L. (1991) A new view of prostaglandin E regulation of the immune response. Immunol. Today 12, 349-352. Poli, G., Bressler, P., Kinter, A., Duh, E., Timmer, W.C., Rabson, A.. Justement, J.S.. Stanley, S. and Fauci, A.S. (1990) Interleukin 6 induces human immunodeficiency virus expression in infected monocytic cells alone and in synergy with tumor necrosis factor a by transcriptional and post-transcriptional mechanisms. J. Exp. Med. 172. 151- 1%

114

D. Miao et al. 1 Journal of Reproductive Imnut~ology 30 (1996) 97- 114

Quayle, A.J., Kelly, R.W., Hargreave, T.B. and James, K. (1989) Immunosuppression by seminal prostaglandins. Clin. Exp. Immunol. 75, 387-391. Rappaport, R.W. and Dodge, G.R. (1982) Prostaglandin E inhibits the production of human interleukin-2. J. Exp. Med. 155, 943-948. Roper, R.L., Conrad, D.H., Brown, D.M., Warner, G.L. and Phipps, R.P. (1990) Prostaglandin E2 promotes IL-4-induced IgE and IgGl synthesis. J. Immunol. 145, 2644-2651. Roper, R.L., Ludlow, J.W. and Phipps, R.P. (1994) Prostaglandin E2 inhibits B lymphocyte activation by a CAMP-dependent mechanism: PGE-inducible regulatory proteins. Cellular Immunol. 154, 296-308. Skibinski, G., Kelly, R.W., Harrison, C.M., McMillan, L.A. and James, K. (1992) Relative immunosuppressive activity of human seminal prostaglandins. J. Reprod. Immunol. 22, 185-195. Skibinski, G., Kelly, R.W. and James, K. (1993) The effect of seminal plasma prostaglandins on T cells and NK cell? Immunomethods 2, 227-236. Tarter, T.H., Cunningham-Ruadles, S. and Koide. S.S. (1986) Suppression of natural killer cell activity by human seminal plasma in vitro: identification of 19-OH PGE as the suppressor factor. J. immunol. 136, 2862-2867. Templeton, A.A., Cooper, I. and Kelly, R.W. (1978) Prostaglandin concentrations in the semen of fertile men. J. Reprod. Fertil. 52, 147-152. Vallely, P.J. and Rees, R.C. (1986) Seminal plasma suppression of human lymphocyte responses in vitro requires the presence of bovine serum factors. Clin. Exp. Immunol. 66, 181-187. Vallely, P.J., Sharrard, R.M. and Rees, R.C. (1988) The identification of factors in seminal plasma responsible for suppression of natural killer cell activity. Immunology 63, 451456. Van der Pouw Kraan, T.C.T.M., Boeije L.C.M., Smeenk, R.J.T., Wijdenes, J. and Aarden, L.A. (1995) Prostaglandin E2 is a potent inhibitor of human interleukin 12 production. J. Exp. Med. 181, 775-779. Vercammen, C. and Ceuppens, J.L. (1987) Prostaglandin E2 inhibits human T cell proliferation after crosslinking of the CDS-Ti complex by directly affecting T cells at an early step of the activation process. Cell. Immunol. 104, 24-36. Watanabe, S., Yssel, H., Harada, Y. and Arai, K. (1994) Effects of prostaglandin Ez on ThO-type human T cell clones: modulation of function of nuclear proteins involved in cytokine production. Int. Immunol. 6, 523-532. Weissman, D., Poli, G. and Fauci, A.S. (1994) Interleukin 10 blocks HIV replication in macrophages by inhibiting the autocrine loop of tumor necrosis factor a and interleukin 6 induction of virus. AIDS Res. Hum. Retrovir. 10, 1199- 1206. Zhong, W.W., Burke, P.A., Drotar, M.E., Chavali, S.R. and Forse, R.A. ( 1995) Effects of prostaglandin E2, cholera toxin and 9-bromocyclic AMP on lipopolysaccharide-induced gene expression of cytokines in human macrophages. Immunology 84, 446-452.