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SISt.XtONS, R.L. & Rt~SSH., P.S. (1962), The antigenicity of mouse trophoblast. Ann. N.Y. Acad. Sci., 99, 717-732. STAR~EV, P.M., SARGENT,I.L. & REDSlAN, C.W.G. (1988), Cell population in human early pregnancy decidua - characterization and isolation of large granular lymphocytes by flow cytome*,ry. Immunology, 65, 129-134. SUNDERLAND,C.A., REDMAN,C.W.G. & STmRAT,G.M. (1981), HLA A, B, C, antigens are expressed on nonviilous trophoblast of the early human placenta. J. ImmunoL, 127, 2614-2615. T.xxvHK, O.W., HUNT, J.S. & WOOD, G.W. (1986a), Implication of prostaglandin E2 in soluble factor-mediated immune suppression by murine decidual cells. Amer. J. Reprod. hnmunol., Microbiol., 12, 111-117. TAWHK, O.W., HUNT, J.S. & WOOD, G.W. (1986b), Partial characterization of uterine cells responsible for suppression of murine maternal anti-foetal immune responses. J. Reprod. Immunol., 9, 213-224. V~TOt~,C.K. & M~:K~:ERJEE,B.B. (1978), Vaccine containing autogenous term placenta and an immunopotentiator to reduce incidence of autochthonous cancer. Brit. J. Cancer., 37, 316-318. Y,XGEL,S., KHOKHA,R., DENHARDT,D.T., KERBEL,R.S., PARHAR,R.S. & LALA, P.K. (1989a), Mechanisms of cellular invasiveness. A comparison of amnion invasion in vitro and metastatic behaviour in vivo. J. nat. Cancer Inst., 81, 768-775. YAt~;EI,S., PARHAR,R.S., JEFFREY,J.J. & LALA,P.K. (1988), Normal non-metastatic human trophoblast cells share in vitro invasive properties of malignant cells. J. Cell Physzol., 136, 455-462. YAGEL, S., WARNER, A.H., NE,-LANS, H.N., LALA, P.K., ~,VAGHORN,C. & DENHARDT,D.T. (1989b), Suppression by cathepsin L inhibitors of Lhe invasion of amnion membranes by murine cancer cells. Cancer Res., 49, 3553-3557. YAMASHIRO,S., BAST,T. & CROY,B.A. (1989), Failure of the beige genotype to alter the morphology of uterine granulated metrial gland cells. Proc. Elect. Microscopy Soc. Am., 47, 908-909. ZUCgERMANN, F.A. & HEAD, ! R . ~1987), Murine trophoblast resists cell-mediated lysis. - I. Resistance to allospecific cytotoxic T lymphocytes. J. ImmunoL, 139, 2856-2864. ZUCKERMANN, F.A. & HEAD, J.R. (1988), Murine trophoblast resists cell-mediated lysis. - 1I. Resistance to natural cell-mediated cytotoxicity. Cell lmmunoL, 16, 274-286.

Supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada.

POSSIBLE

MEDIATORS

IN ENDOTOXIN-INDUCED

ABORTION

M. Parant

Laboratory o f lmmunopharmacology, C N R S UPR 405, 15, rue de l'Ecole de M~decine, 75270 Paris Cedex 06

Introduction.

Bacterial endotoxins (LPS; lipopolysaccharides) have been suggested to be an important causative agent o f

perinatal morbidity, since the first observation m 1944 by Zahl and Bjerknes that administration into pregnant animals was accompanied by foetal death or expulsive abortion. Histologic

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ABORTION

examination of the placenta showed patterns of ischemic coagulation necrosis (Mc Kay and Wong, 1963), supporting early observations on similarities between placental injury and haemorrhagic necrosis of grafted tumours produced by LPS injection (Zahl and Bjerknes, 1944). The inflammatory and thrombotic reactions elicited by LPS have since been extensively documented and it has been proposed that cytokines are the mediators of these phenomena (Cybulsky et al., 1988). Actually, many aspects of the inflammatory response to invasive organisms appear to be governed by polypeptides, in turn produced by immune effector cells (Beutler and Cerami, 1989). However, in the abortifacient effect of LPS, the role of other host-derived intermediary molecules such as serotonin (5-HT), prostaglandins or platelet-activating factor (PAF) would deserve additional studies either as a direct response to the bacterial agent or as a cytokine-mediated consequence. Endotoxin-induced abortion.

Infection-associated preterm labour has been related to the presence of LPS "~'-'tmainitiates a series of events leading to the rupture of foetal membranes (Casey et al., 1989). In several animal species, induction of abortion was regularly obtained with doses that did not kill the pregnant female (Zahl and Bjerknes, 1944; Takeda and Tsuchiya, 1953; Rieder and Thomas, 1960). Moreover, pretreatment with glucocorticoids, known to exert strong protection against the lethal effect of LPS, did not prevent abortion (Rieder and Thomas, 1960; Parant and Chedid, 1964). Foetal mortality occurred a few hours after intravenous injection of LPS into the female (Darrieulat and Parant, 1971), but did not appear to be primarily responsible for abortion (Zahl and Bjerknes, 1944). LPS did not cause death when injected directly into the foetus (Parant and Chedid, 1964), suggesting that it induces abortion by its action on the mother. Involvement of an endocrine mechanism (ovary alteration

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or ocytocin secretion) was excluded, since mouse pregnancy, maintained by progesterone treatment after ovariectomy or hypophysectomy, was interrupted at the same doses of LPS (Chedid et al., 1962). Thrombosis of the placental and uterine vasculature was considered to be a major effect of LPS, responsible for much of the ultimate necrosis (McKay and Wong, 1963). Prevention abortion.

of

endotoxin-induced

Several attempts have been made to prevent abortion in animals given LPS, leading to possible prophylactic procedures a n d / o r to an improved understanding of the mechanisms. Results suggest that the abortifacient effect may be dissociated from toxic shock. 3hus, passive tranfer of specific antibodies raised against LPS or homologous bacteria protected pregnant mice from abortion but were poorly active against lethality (Darrieulat et al., 1978). As already mentioned, glucocorticoids were ineffective in preventing abortion, although they exhibited a considerable degree of protection against endotoxic shock, even in pregnant mice which are slightly more sensitive to the LPS effect (Parant and Chedid, 1964). The release of vasoactive substances such as histamine or serotonin from platelets and other sources may contribute to the syndrome of endotoxic shock and favour the development of intravascular thrombosis. The protective effect of chlorpromazine against the lethal effect of LPS has been related to the prevention of lesions caused by vasoactive materials. In pregnant mice, chlorpromazine was shown to prevent the effect of LPS (Parant and Chedid, 1964) and also serotonin-induced intrauterine foetal death related to vasoconstriction of the maternal vessels (Lindsay and Poulson, 1963). Then an antagonist of serotonin, methysergide, was used to counteract the abortifacient effect of LPS or of 5-HT (Darrieulat and Parant, 1971). The possible involvement of other secondary mediators has

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becn postulated. PAF is released during endotoxaemia. By itself it did not alter the development of pregnancy in mice, but PAF antagonists decreased foetal mortality elicited by LPS (Etienne et al., 1988). Because of the effect of LPS on monocytes/macrophages in causing the production of prostaglandins that are mediators of myometrial contractions, inhibitors of their synthesis were successfully used in pregnant animals to prevent abortion (Skarnes and Harper, 1972). It has also been reported that, after treatment of decidual explants with LPS, the production of PGF2a increased (Casey et al., 1989). It has been known for many years that LPS can elicit both local and systemic microvascuiar alterations. Available evidence suggests that intcrleukin-1 (IL-1) and tumour necrosis factor (TNF) are the principal intrinsic mediators of these LPS-induced reactions (Cybulsky etal., 1988). Haemorrhagic necrosis of tumours elicited by LPS was soon attributable to the production of an endogenous protein (TNF) released in response to LPS. Later work supported the notion that TNF is an important mediator of endotoxicity (reviewed in Beutler and Cerami, 1989). Since LPS is also a potent stimulator of IL-1 production, a b y t u ~ t , , t c t t t i a t blli;l,I e~5 O l O l O g l C a l a c t i v i t i e s

with TNF, both mediators could be involved in LPS-induced abortion, producing placental hacmorrhage through the induction of procoagulant activity.

involvement of cytokines foetoplacental injury.

in

Cytokines have been implicated in premature pregnancy termination, mainly because uteroplacental macrophages can be activated by bacterial LPS (reviewed in Hunt, 1989). Cord blood monocytes, which did not spontaneously release cytokines, responded to LPS stimulation in producing biologically active IL-1 and TNF (Weathe~'stone and Rich, 1989). Human decidual explants incubated with LPS re!ea~ed IL-1 (Romero etal., 1989b) and TNF (Casey et al., 1989). Moreover,

TNF has been identified in human amniotic fluid and TNF receptors have been characterized in the villous tissue of human placenta (in Hunt, 1989). TNF has been proposed as an important mediator in the pathology of malaria and could be responsible for foetal loss seen in this disease, as shown in mice infected with the parasite (Clark and Chaudhri, 1988). Administration of recombinant human IL-1 or TNF on day 12 of gestation in rats caused placental necrosis and foetus resorption when checked one week later. Their effect appeared less drastic than that prcduced by LPS (Silen et al., 1989). In mice, under the experimental conditions used with LPS, a single injection of recombinant human TNF between 12 and 16 days of pregnancy caused foetal death, stillbirth and sometimes premature delivery within 24 h (Parant, 1987). Recombinant murine TNF produced abortion in mice at the same doses (Parant et al., 1990). Human IL-1 was almost as effective as TNF, also inducing vaginal ble~:ding, foetal death and necrosis of the decidua. With the dose of cytokines that killed all foetuses, no mortality occurred in the treated females (unpublished data). Following injection of LPS (1 ~g) into pregnant mice, a sharp rise in circulating TNF was observed showing a pattern of response similar ~o that produced in controls; the level of TNF released in the blood was equal to or sometimes higher than that during pregnancy. Of interest is that pretreatment with glucocorticoids inhibited LPS-induced production of TNF as effectively in pregnant and in control animals. The hormone is known to exert a blocking effect on IL-1 or TNF production by monocytes/macrophages. A preliminary evaluation of serum interleukin-6 (IL-6) in pregnant and control mice given LPS has shown the production of similar levels that were not markedly modified by pretreatment with glucocorticoids (unpublished data). Circulating IL-6 was first demonstrated in mice receiving an LPS

DOES

IMMUNE

ABORTION

injection (Coulie et al., 1987). IL-6 may have an important role in inflammatory responses, can manifest a broad spectrum of diverse biological responses and has been linked specifically to the stimulation of acute phase plasma protein synthesis by hepatocytes. IL-1 and TNF share the ability to stimulate endothelial cells for production of IL-6 (Shalaby et al., 1989). Therefore, a possible role of IL-6 in LPS- or in cytokine-induced abortion is to be considered. It is tempting to speculate on the role of IL-6 detected in human amniofic fluM and associated with intraamniotic infection. Moreover, decidual tissue explants released high levels of IL-6 when stimulated with LPS (Romero et al., 1989a).

Possible intermediary effector molecules. In studies of LPS-induced abortion performed before the role of cytokines was considered, the role of secondary mediators such as 5-HT or prostaglandins was suggested on the basis of their direct effect on pregnancy, the protective action of antagonists and their potential production in response to LPS stimulation. Serotonin is likely to be released from aggregated platelets in capillaries, whereas synthesis and secretion of .iJiuStctE,[~llUltl:~ . . . . . ' - - " : - - o r o f PAF may be a response to LPS stimulation of endothelial cells and macrophages. Endothelium is a target tissue for a direct action of LPS and is responsible for the induction of procoagulant activity and the suppression of anticoagulant

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mechanisms (Cybulsky et al., 1988). Moreover, the influence of such secondary effector molecules, alone or in synergy, cannot be excluded in the effect of cytokines on the outcome of pregnancy. Interactions of cytokines and vascular endothelial cells and the ensuing biological events have received much attention in recent years. Both IL-1 and TNF stimulate endothelial cells and induce different but partially overlapping responses, resulting in a procoagulant activity. The cytokines also stimulate these cells to synthesize prostaglandins which may further aggravate the inflammatory reaction (Pober et al., 1988). Furthermore, TNF and, to a lesser degree IL-1, are potent inducers of synthesis and release of PAF by various cell types, and their activity is elevated on endothelial cells (Bussolino et aL, 1988).

Conclusion. A multiplicity of inflammatory agents appear to be involved directly or indirectly in placental injury and foetal death. In LPS-induced abortion, the absence of any protective effect of glucocorticoids indicates that the production of TNF and/or IL-1 may not be a prerequisite in the mechanisms, although it is not known whether the hormone can locally block cytokine production by uteroplacental macrophages. However, the abortifacient effect of LPS, IL-I and TNF suggests that they may act, at least partially, through common pathways.

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host response. Ann. Rev. hnmunol., 7, 625-655. BUSSOHNO,F., CAMUSSLG. & BAOIONI,C. (1988), Synthesis and release of platelet-activating factor by human vascular endothelial cells treated with tumor necrosis factor or interleukin 10(. J. biol. Chem., 263, 11856-11861. CasEv, M.L., Cox, S.M., BEUTLER,B., MILEWICH,L. & MACDONALD,P.C. (1989), Cachectin/tumor necrosis factor-a formation in human decidua. J. clin. Invcst., 83,430-436. CHeDID, L., BORER,F. & PArANt, M. (1962), Etude de l'action abortive des endotoxines inject6es h la souris gravide normale, castr6e ou hypophysectomis~e.Ann. Inst. Pasteur, 102, 77-84. CLARK,I.A. & CHAUDrl,G. (1988), Tumor necrosis factor in malaria-inducedabortion. Amer. J. Med. Hyg., 39, 246-249.

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C~L'I I~, P.G., CAYPHAS,S., VINK, A., UYTTENHOVE,C. • VAN SNICK, J. (1987), Interleukin. . . . . . . ;~,~n h,,h~;n,,,~ and plasmacytoma growth factors induced by lipopolysaccharide in vivo. Europ. J. lmmunol., 17, 1217-1220. CY~utsr~v, M.I., CHAN, M.K.W. & MOVAT, H.Z. (1988), Biology of disease. Acute inflammation and microthrombosis induced bv endotoxin, interleukin 1 and tumor necrosis factor and their implication in Gram'-negative infection. Lab. Invest., 58, 365-378. DARRIEULAT, F. & PARAN't, M. (1971), Action d'un antagoniste de la s6rotonine, le m6thysergide, sur l'effet abortif ou 16tal des endotoxines bact6riennes chez la souris. Ann. Inst. Pasteur, 121,665-673. DA~ktEULAT, F., P~RAN-f,M. & CHEDID, L. (1978), Prevention of endotoxin-induced abortion bv trcatmcnt of mice with antisera. J. infect. Dis., 137, 7-12 Des Pm:z, P?,.M., Hol,eWqTZ, H.I. & HooK, E.W. (1961), Effects of bacterial endotoxin on rabbit platelcts.-l. Platelet aggregation and release of platelet factors in vitro. J. exp. Med., 114, 857-873. ETIENNE, A., HECQUET,F. & BRAQUET,P. (1988), Possible involvement of platelet-activating factor in endotoxin-induced abortion, in "Bacterial endotoxins: pathophysiological effects, clinical significance, and pharmacological control" (J. Levin, H.R. Biiller, J.W. ten Cate, S.J.H. van Deventer & A. Sturk) (pp. 135-143). Alan R. Liss, New York. HUNa-, J.S. (1989), Cytokine networks in the uteroplacental unit: macrophages as pivotal regulatory cells. J. Reprod. lmmunol., 16, 1-17. L'NDS',V, D., PoulsON, E. & ROBSON, J.M. (1963) The effect of 5-hydroxyryptamine on pregnancy. J. Endoerin., 26, 85-96. MAND.,~, T., NtSHIGA~t, F., MORI, J. & SmMoMorm, K. (1988), Important role of serotonin in the antitumor effects of recombinant human tumor necrosis factor-