Antibody-Induced Pregnancy Loss

TNF-α Is a Critical Effector and a Target for Therapy in Antiphospholipid Antibody-Induced Pregnancy Loss

This information is current as Jessica Berman, Guillermina Girardi and Jane E. Salmon of September 24, 2021. J Immunol 2005; 174:485-490; ; doi: 10.4049/jimmunol.174.1.485 http://www.jimmunol.org/content/174/1/485 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TNF-␣ Is a Critical Effector and a Target for Therapy in Antiphospholipid Antibody-Induced Pregnancy Loss1

Jessica Berman, Guillermina Girardi, and Jane E. Salmon2

The antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, intrauterine growth restriction, and vascular thrombosis in the presence of antiphospholipid (aPL) Abs. Our studies in a murine model of APS induced by passive transfer of human aPL Abs have shown that activation of complement and recruitment of neutrophils into decidua are required for fetal loss, and emphasize the importance of inflammation in aPL Ab-induced pregnancy loss. In this study, we examine the role of TNF-␣ in pregnancy complications associated with aPL Abs in a murine model of APS. We show that aPL Abs are specifically targeted to decidual tissue and cause a rapid increase in decidual and systemic TNF-␣ levels. We identify the release of TNF-␣ as a critical intermediate that acts downstream of C5 activation, based on the fetal protective effects of TNF-␣ deficiency and TNF blockade ␣ ␣ and on the absence of increased TNF- levels in C5-deficient mice treated with aPL Abs. Our results suggest that TNF- links Downloaded from pathogenic aPL Abs to fetal damage and identify TNF blockade as a potential therapy for the pregnancy complications of APS. The Journal of Immunology, 2005, 174: 485–490.

he antiphospholipid syndrome (APS)3 is characterized by Evidence from human pregnancy studies points to a strong as- arterial and venous thromboses and pregnancy loss that sociation between maternal Th2-type immunity and successful occur in the presence of antiphospholipid (aPL) Abs (1). pregnancy, whereas Th1-type immune reactivity is associated with T http://www.jimmunol.org/ APS is common in patients with systemic lupus erythematous, and pregnancy loss (5). Mitogen-stimulated PBMC, harvested at the it is also found in individuals without other autoimmune features. end of the first trimester, have consistently been shown to produce Although it is clear that the specific antigenic reactivity of aPL Abs higher levels of IL-4, IL-5, IL-6, and IL-10 in patients who went and their targeting to decidual tissue are critical to their effect, the on to have normal pregnancies than in those who had recurrent pathogenic mechanisms that result in fetal injury in vivo are in- spontaneous miscarriages. In contrast, levels of TNF-␣, IL-2, and completely understood. IFN-␥ were uniformly higher in patients who subsequently had Therapy for pregnant women with APS is focused on preventing miscarriages (6, 7). Indeed, the preponderance of Th2 cytokines thrombosis, but anticoagulation is only partially successful in relative to Th1 cytokines in the local milieu of the fetus is con- averting miscarriage and carries risks for fetus and mother. Al- sidered to be essential for its survival. The proinflammatory Th1- by guest on September 24, 2021 though experimental models have emphasized the role of throm- dominant response that underlies clinical pregnancy failure is de- bosis in placental tissue, histopathologic findings in placentas from pendent on immunologic factors that may be amplified by women with APS argue that proinflammatory factors may contrib- environmental stimuli, such as LPS, autoantibodies, including aPL ute to injury (2, 3). Our recent studies showing that activation of Abs, and stress (8, 9). Importantly, TNF-␣ is sensitive to modu- complement and recruitment of neutrophils into decidua are re- lation by such environmental factors. quired for fetal loss in a murine model of APS underscore the TNF-␣ is a multifunctional Th1 cytokine with roles in regulat- importance of inflammation in aPL Ab-induced pregnancy loss (4). ing hormone synthesis, placental architecture, and embryonic de- However, the specific effectors of fetal injury remain unknown. velopment (10), and elevated TNF-␣ levels are associated with Because TNF-␣ is released when infiltrating inflammatory cells miscarriage (11) and pre-eclampsia (12). Patients with recurrent are activated by complement split products and elevated levels of pregnancy loss, both with and without aPL Abs, evidence enrich- TNF-␣ have been associated with pregnancy failures, we consid- ment of TNF-␣ promoter alleles associated with increased cyto- ered the possibility that TNF-␣ may have a role in pregnancy com- kine levels (11, 13). Increased placental levels of TNF-␣ have been plications associated with APS. associated with pregnancy failure in mice (14), administration of TNF-␣ increases abortion rates (6), and blockade of TNF-␣ has been shown to prevent stress-induced miscarriage in murine mod- Department of Medicine, Hospital for Special Surgery-Weill Medical College of Cor- els of abortion (15). That TNF-␣ can directly promote tissue dam- nell University, New York, NY 10021 age in pregnancy has been suggested by in vitro studies in which Received for publication July 8, 2004. Accepted for publication October 4, 2004. TNF-␣-activated maternal monocytes bind to LFA-1 on placental The costs of publication of this article were defrayed in part by the payment of page syncytiotrophoblasts and induce apoptosis (16). charges. This article must therefore be hereby marked advertisement in accordance In this study, we examine the role of TNF-␣ in aPL Ab-induced with 18 U.S.C. Section 1734 solely to indicate this fact. fetal injury in a murine model of APS in which pregnant mice 1 This work was supported in part by a grant from the National Institutes of Health (AI055007) and by the Alliance for Lupus Research and the Mary Kirkland Center for receive human IgG containing aPL Abs. Passive transfer of IgG Lupus Research at Hospital for Special Surgery. from women with recurrent miscarriage and aPL Abs results in 2 Address correspondence and reprint requests to Dr. Jane E. Salmon, Hospital for fetal loss and growth restriction (17). The frequency of fetal re- Special Surgery, 535 East 70th Street, New York, NY 10021. E-mail address: sorption in aPL Ab-treated mice is ϳ40% compared with Ͻ10% in [email protected] mice treated with IgG from healthy individuals, and the average 3 Abbreviations used in this paper: APS, antiphospholipid syndrome; aPL, antiphospholipid; aPL-IgG, human IgG containing aPL Abs; NH-IgG, normal human IgG; weight of surviving aPL Ab-treated fetuses is reduced by 50% (4, PEG, polyethylene glycol; sTNFRI, soluble TNF-␣R type I; aCL, anticardiolipin. 13). We have shown that activation of complement, specifically

C5a-C5a receptor interactions, is a necessary intermediary step for Statistical analyses these clinically relevant deleterious effects of aPL Abs (4). How- Data are expressed as mean Ϯ SD. Student’s t test was used to compare ever, the downstream pathogenic mediators of placental and fetal fetal resorption rates and fetal weights between groups. A probability of damage induced by aPL Abs and the sites and agents for optimal Ͻ0.05 was used to reject the null hypothesis. therapeutic interventions to prevent APS are not yet clear. In this study, we test the hypotheses that aPL Abs targeted to decidual Results tissue lead to the release of TNF-␣, that TNF-␣ is a critical me- APL Abs localize to decidual tissue in pregnant mice diator in aPL Ab-induced damage, and that blockade of TNF-␣ We have previously shown that passive transfer of human IgG might be an effective treatment to prevent pregnancy loss. from APS patients into pregnant mice causes inflammation within deciduae and fetal resorption or growth restriction (4, 13). These in Materials and Methods vivo studies demonstrated a direct pathogenic role for aPL Abs. In vitro studies in human and murine placentas have shown that tro- Mice phoblast cell membranes are targets for ␤2GPI-dependent and Adult mice (6–8 wk) were used in all experiments. BALB/c mice were ␤2GPI-independent aPL Abs (23), suggesting that these Abs are purchased from Taconic Farms. TNF-␣-deficient, TNFtm1Gkl/J (TNFϪ/Ϫ) ϩ ϩ specifically targeted to the placenta. In this study, we investigated mice and TNF / background strain (B6129S6) were obtained from The Jackson Laboratory (18). C5-deficient mice (C5Ϫ/Ϫ) (B10.D2-H2dH2-T18c the tissue localization and kinetics of aPL Ab handling in an in Hco/o2SnJ) and C5-sufficient mice (C5ϩ/ϩ) (C57BL/10SnJ) were also ob- vivo model. BALB/c mice were treated with human aPL-IgG (10 tained from The Jackson Laboratory (19, 20). Procedures that involved mg, i.p.) or NH-IgG on day 8 of pregnancy, and the presence of mice were approved by the local Committee on Animal Use in Research human IgG was assessed in tissue by immunohistochemistry. We Downloaded from and Education and were conducted in strict accordance with Guidelines for the Care and Use of Laboratory Research Animals promulgated by the detected human IgG in mouse deciduae within 30 min after i.p. National Institutes of Health. injection of aPL-IgG into pregnant BALB/c mice (Fig. 1a). Anal- ysis of kidney, lung, and liver sections from these animals showed Preparation of aPL and other Abs no evidence of human IgG deposition (data not shown). In deciduae of mice treated with IgG from healthy individuals, there was Human IgG containing aPL Abs (aPL-IgG) was obtained from patients with APS (characterized by high-titer aPL Abs (Ͼ140 GPL U), thrombo- no detectable human IgG deposition (Fig. 1b). To demonstrate that http://www.jimmunol.org/ ses, and/or pregnancy losses) (1). Normal human IgG (NH-IgG) was Abs reactive with aPL, rather than other autoantibodies or xeno- obtained from healthy non-autoimmune individuals. IgG was purified by reactive Abs that may be present in polyclonal human IgG, are affinity chromatography using protein G-Sepharose chromatography col- deposited in deciduae, we treated pregnant mice with human aPL umns (Amersham Biosciences) as previously described (13). The genera- mAb (100 ␮g, i.v.). The results of immunohistochemical studies of tion, structure, and specificity of the human IgG1 aPL mAb (mAb 519) was previously described (21). All IgG samples were treated to deplete endo- decidual tissue from mice treated with human aPL mAb were sim- toxin with Centriprep ultracentrifugation devices (Millipore) and deter- ilar to those obtained with polyclonal aPL-IgG (Fig. 1c). mined to be free of endotoxin using the Limulus amebocyte lysate assay. To determine whether aPL-reactive Abs are specifically targeted to decidual tissue, we compared the handling of aPL-IgG in preg- Murine pregnancy model nant mice with that in nonpregnant mice. Analysis of serial blood by guest on September 24, 2021 Female mice were mated with previously isolated males, and the presence samples obtained after injection of aPL-IgG showed that, whereas of a vaginal plug was defined as day 0 of pregnancy. On days 8 and 12 of levels of total human IgG in the circulation did not differ between pregnancy, mice were treated with i.p. injections of aPL-IgG (10 mg) or pregnant and nonpregnant mice (Fig. 1d), titers of human aCL- NH-IgG (10 mg). In some studies, mice were treated on days 8 and 12 of reactive IgG were persistently and significantly lower in pregnant pregnancy with i.v. injections of human aPL mAb (100 ␮g) (21). Mice were sacrificed on day 15, uteri were dissected, fetuses and placentas were mice (e). The plasma volume of pregnant mice does not increase weighed, and fetal resorption frequency was calculated (number of resorp- significantly by day 8, as measured by Evans blue dye dilution tions divided by the total number of formed fetuses and resorptions). Re- (pregnant vs nonpregnant, 1.07 Ϯ 0.06 vs 1.10 Ϯ 0.08 ml); there- sorption sites are easily identified and result from the loss of a previously fore, the decreased levels of aCL-reactive IgG cannot be explained viable fetus at that site. To inhibit TNF-␣, BALB/c mice were treated with polyethylene glycol (PEG)-conjugated soluble TNF-␣R type I (PEG by a dilutional effect (24). Rather, our results suggest that aPL Abs sTNFRI; Amgen) on days 8, 10, and 12 of pregnancy (5 mg/kg, i.p). Mice are selectively targeted and bound to decidual tissues in pregnant received PEG sTNFRI 1 h before aPL-IgG injections on days 8 and 12. To mice, and thereby removed from the circulation. inhibit C5, mice were treated on days 8 and 10 with anti-C5 mAb (1 mg, i.p.) (22) or murine IgG, as a control. Treatment with aPL Abs causes a rapid increase in decidual and systemic TNF-␣ ␣ Detection of plasma levels of Abs and TNF- To determine whether deposition of aPL-IgG in deciduae causes Mice were bled on day 8 of pregnancy 30, 60, and 90 min, and 2, 4, 6, 8, local TNF-␣ release, we performed immunohistochemical studies 10, 12, 18, 24, 30, 36, and 42 h after administration of aPL-IgG or NH-IgG of decidual tissues from day 8 of pregnancy removed 30, 60, 90, injection. Anticardiolipin (aCL) activity was measured by an ELISA ac- and 120 min after the i.p. injection of aPL-IgG. Intense staining for cording to the manufacturer’s instructions (Sigma-Aldrich). Levels of hu- ␣ man IgG and mouse TNF-␣ were measured by standard ELISA methods TNF- was evident by 30 min after administration of aPL-IgG (Zeptometrix and OptEIA Pharmingen, respectively). (Fig. 2a) and persisted through 120 min. The time course for the appearance of TNF-␣ in decidual tissue was similar to that of Immunohistochemistry human IgG deposition. Immunohistochemistry studies indicated that trophoblasts, as well as infiltrating inflammatory cells, were Deciduae were removed from mice on day 8 of pregnancy, 30, 60, 90, and ␣ ␣ 120 min after administration of aPL-IgG or NH-IgG, frozen in OCT com- producing TNF- (Fig. 2b). There was no TNF- in deciduae of pound, and cut into 10-␮m sections. After quenching endogenous perox- mice treated with NH-IgG (Fig. 2, c and d). idase with 1% H2O2 in methanol and blocking nonspecific binding sites The administration of aPL-IgG to pregnant mice also caused a with normal goat serum (Cappel), sections were incubated with goat anti- rapid and sustained increase in serum concentrations of TNF-␣. mouse TNF-␣, goat anti-human IgG (Sigma-Aldrich), or goat anti-mouse C3 (Cappel), followed by anti-goat IgG conjugated to HRP (Sigma-Al- Within 20 min after i.p. injection of aPL-IgG, circulating levels of drich). Bound HRP was detected with diaminobenzidine. Sections were TNF-␣ increased dramatically, peak levels occurred at 60 min, and counterstained with hematoxylin. the elevation of TNF-␣ persisted for Ͼ8 h (Fig. 2e). Importantly, The Journal of Immunology 487 Downloaded from

FIGURE 1. aPL Abs are targeted to the decidua. a–c, BALB/c mice were treated with aPL-IgG (10 mg, i.p.), NH-IgG (10 mg, i.p.), or human aPL mAb (100 ␮g, i.v.) on day 8 of pregnancy (n ϭ 6–8 mice/group). Mice were sacriﬁced 60 min after injections, and histologic sections of deciduae were stained with anti-human IgG Abs. In mice treated with polyclonal aPL-IgG (a) or human aPL mAb (c), human IgG was present throughout decidual tissue. In http://www.jimmunol.org/ contrast, there was minimal IgG deposition in mice treated with NH-IgG (b). d and e, Pregnant mice (on day 8) and nonpregnant mice were treated with aPL-IgG (n ϭ 5), and serial blood samples were collected and analyzed for plasma levels of human IgG and aCL reactivity. Levels of human IgG (d) and aCL Abs (e) increased to a similar extent in pregnant and nonpregnant mice. Although total human IgG levels were not different in pregnant and nonpregnant mice, aCL titers fell more rapidly in pregnant mice (p Ͻ 0.01), suggesting that aCL-reactive human IgG is speciﬁcally removed from circulation.

TNF-␣ levels were not increased in pregnant mice that received sTNFRI on days 8, 10, and 12, fetal resorption frequency was not NH-IgG or in nonpregnant mice treated with aPL-IgG (Fig. 2e). increased by administration of aPL-IgG (aPL-IgG vs NH-IgG, by guest on September 24, 2021 Taken together, our results show a temporal relationship be- 21 Ϯ 4vs19Ϯ 6%) (Fig. 3b). Although there was modest increase tween binding of human IgG to deciduae, local expression of in fetal loss associated with PEG sTNFRI in mice treated with TNF-␣, and elevation of systemic TNF-␣. That the increase in NH-IgG (NH-IgG plus PEG sTNFRI vs NH-IgG, 19 Ϯ 6vs8Ϯ circulating TNF-␣ was evident only in pregnant mice treated 6%; p Ͻ 0.02), blockade of TNF-␣ in aPL Ab-treated mice de- with aPL-IgG suggests that aPL Abs localized to deciduae ini- creased pregnancy loss by 50% (aPL-IgG vs aPL-IgG plus PEG tiate the production of proinflammatory cytokines by tropho- sTNFRI, 42 Ϯ 6vs21Ϯ 4%; p Ͻ 0.005) (Fig. 3b). The magnitude blasts and leukocytes. of the improvement in fetal survival was comparable to that observed in TNFϪ/Ϫ mice (Fig. 3a). Blockade of TNF-␣ improves pregnancy outcomes in mice treated with aPL Abs Activation of complement is required for TNF-␣ release induced To directly test whether elevated local or systemic TNF-␣ con- by aPL Abs tributes to aPL Ab-induced pregnancy loss, we studied mice with We have previously identified complement component C5 and, a targeted deletion of TNF-␣. If TNF-␣ is a key intermediate in particularly, its cleavage product, C5a, as key mediators of fetal fetal injury, then TNFϪ/Ϫ mice should be protected from preg- injury caused by aPL Abs, and have shown that C5Ϫ/Ϫ mice are nancy loss. In the TNFϩ/ϩ background strain, aPL-IgG caused a completely protected from aPL Ab-induced fetal resorption and Ͼ4-fold increase in the frequency of fetal resorption compared growth restriction (4). C5a attracts and activates neutrophils, with treatment with NH-IgG (43 Ϯ 9vs10Ϯ 5%; p Ͻ 0.001). In monocytes, and mast cells, and stimulates the release of inflam- mice lacking TNF-␣, the harmful effect of aPL Abs on pregnancy matory mediators, including TNF-␣. To determine whether there is outcome was markedly reduced. In fact, the frequency of fetal a relationship between activation of complement, release of resorption in TNFϪ/Ϫ mice that received aPL-IgG was not signif- TNF-␣, and pregnancy loss caused by aPL Abs, we studied C5- icantly different from that of mice treated with IgG from healthy deficient mice. If TNF-␣ is a critical effector of fetal injury that individuals (aPL-IgG vs NH-IgG, 24 Ϯ 4vs17Ϯ 9%; p ϭ NS), acts downstream of C5, we would expect to find no elevation of but it was significantly lower than that observed in TNFϩ/ϩ mice TNF-␣ in C5-deficient mice treated with aPL-IgG. In the C5ϩ/ϩ treated with aPL-IgG ( p Ͻ 0.02) (Fig. 3a). background strain, there was a rapid and sustained increase in cir- As an alternate approach to test the hypothesis that TNF-␣ is a culating TNF-␣ in response to aPL-IgG Abs (Fig. 4a), comparable mediator of aPL Ab-induced pregnancy complications, we blocked to that seen in BALB/c mice (Fig. 2c). In contrast, in C5Ϫ/Ϫ mice, TNF-␣ activity with high-affinity monomeric PEGylated type I there was no increase in TNF-␣ after aPL-IgG; levels were similar TNFR. PEG sTNFRI has been shown to have potent in vivo anti- to those in mice treated with NH-IgG (Fig. 4a). inflammatory activity in a number of murine models of rheumatoid Immunohistochemical studies of decidual tissues from mice arthritis (25, 26). In pregnant BALB/c mice treated with PEG lacking C5 documented the absence of local TNF-␣ release in 488 TNF-␣ IN aPL Ab-MEDIATED PREGNANCY LOSS Downloaded from http://www.jimmunol.org/ FIGURE 3. TNF-␣ deficiency and TNF-␣ blockade protect mice from aPL Ab-induced fetal loss. a, TNFϩ/ϩ and TNFϪ/Ϫ mice were treated with aPL-IgG (10 mg, i.p.) or NH-IgG (10 mg, i.p.) on days 8 and 12 of preg- FIGURE 2. Treatment with aPL Abs causes increased TNF-␣ in decid- nancy. Fetal resorption frequency was determined on day 15 of pregnancy uae and in plasma. a–d, BALB/c mice were treated with aPL-IgG (10 mg, (n ϭ 5–8 mice/group). In TNFϩ/ϩ mice, aPL-IgG induced a Ͼ4-fold in- i.p.) or NH-IgG (10 mg, i.p.) on day 8 of pregnancy. Mice were sacrificed ,ء) crease in the frequency of fetal resorptions compared with NH-IgG 60 min after injections, and histologic sections of deciduae were stained aPL-IgG vs NH-IgG, p Ͻ 0.001). In contrast, in TNFϪ/Ϫ mice, aPL-IgG with anti-mouse TNF-␣ Ab. a and b, In representative sections of decidual did not cause an increase in fetal resorption frequency (aPL-IgG vs NH- tissue from mice treated with aPL-IgG, there was extensive intense staining IgG, p ϭ NS; aPL-IgG, TNFϩ/ϩ vs TNFϪ/Ϫ, p Ͻ 0.02). b, Pregnant for TNF-␣ throughout decidual tissue and surrounding the necrotic em- by guest on September 24, 2021 BALB/c mice were treated with aPL-IgG or NH-IgG on days 8 and 12 of and leukocytes (arrows) expressed (ء) bryonic debris (ED). Trophoblasts pregnancy, and some from each group also received PEG-sTNFRI (5 mg/ TNF-␣. c and d, In mice treated with NH-IgG, there was minimal TNF-␣ kg, i.p) on days 8, 10, and 12 of pregnancy (n ϭ 5–8 mice/group). Ad- present, and the developing embryo (E) was intact. e, Pregnant and non- ministration of PEG-sTNFRI along with aPL-IgG reduced pregnancy pregnant mice (n ϭ 5–7 mice/group) were treated with aPL-IgG or NH- aPL-IgG vs aPL-IgG plus sTNFRI, p Ͻ 0.005), but PEG ,ء) losses by 50% IgG on day 8, and serial blood samples were collected at 0, 10, 20, 30, 60, sTNFRI caused a modest in increased fetal loss in mice treated with NH- 90, 120, 240, 360, and 480 min after injections and assayed for levels of IgG (NH-IgG plus PEG sTNFRI vs NH-IgG, p Ͻ 0.02). TNF-␣. In pregnant mice treated with aPL-IgG, there was a significant increase in plasma TNF-␣ at 20 min (p Ͻ 0.001) after treatment, which peaked at 60 min and persisted for 8 h. aPL-IgG did not cause an increase in TNF-␣ levels in nonpregnant mice. Levels were similar to those in mice activation and links pathogenic aPL Abs to fetal damage. Our con- treated with NH-IgG. clusions are based on the fetal protective effects of TNF-␣ deficiency and TNF blockade and on the absence of increased TNF-␣ levels in C5-deficient mice treated with aPL Abs. Our results iden- response to aPL-IgG. There was extensive staining for TNF-␣ in tify TNF blockade as potential therapy for the pregnancy compli- ϩ ϩ deciduae of C5 / mice that had received aPL-IgG (Fig. 4b), but cations of APS. Ϫ Ϫ only minimal TNF-␣ in C5 / mice (c). Similarly, blockade of C5 Although APS is a systemic disease, its most common clinical cleavage with anti-C5 mAb, which we have previously shown to manifestation is miscarriage. In fact, APS has emerged as a leading completely protect pregnancies from aPL Ab-induced injury (4), cause of pregnancy loss and pregnancy-related morbidity, includ- also prevented local TNF-␣ release (Fig. 4d). Similar results were ing intrauterine growth restriction, premature births, and pre- obtained in mice treated with a C5a receptor antagonist peptide eclampsia (27). Up to 20% of women with recurrent miscarriage (data not shown). Collectively, our data indicate that TNF-␣,a have aPL Abs, and in ϳ15% of otherwise apparently normal mediator of pregnancy loss, is produced in response to comple- women, aPL Abs are the sole explanation for recurrent fetal loss ment activation by aPL Abs. (28, 29). That aPL Abs are specifically targeted to trophoblasts is consistent with these clinical observations. During trophoblast dif- Discussion ferentiation, phosphatidylserine is externalized on the trophoblast In this paper, we report experiments in a murine model of APS outer leaflet where it provides a target for aPL Abs (27, 30). Our induced by passive transfer of human aPL Abs that show that aPL in vivo studies show that aPL Abs are selectively removed from Abs are preferentially bound by decidual tissues where they induce the circulation in pregnant mice and specifically bound to decidual local complement activation, production of inflammatory media- tissues where they activate complement via the classical pathway tors, and ultimately pregnancy loss. We have identified the release (4). Effectors downstream of complement activation, such as of TNF-␣ as a critical intermediate that acts downstream of C5 TNF-␣, provide a means by which these Abs cause pregnancy The Journal of Immunology 489

FIGURE 4. APL Ab-induced increases in TNF-␣ are downstream of complement activation. a,C5ϩ/ϩ and C5Ϫ/Ϫ mice were treated with aPL-IgG (10 mg, i.p) or NH-IgG (10 mg, i.p.) on day 8 of pregnancy, and serial blood samples were collected and analyzed for TNF-␣ levels (n ϭ 5–8 mice/group). In C5ϩ/ϩ mice, TNF-␣ levels increased rapidly and remained elevated after administration of aPL-IgG (p Ͻ 0.005), whereas in C5Ϫ/Ϫ mice there was no change in TNF-␣ after treatment with aPL-IgG. TNF-␣ levels did not change in mice that received NH-IgG. b–d, Decidual tissue from mice sacriﬁced 60 min after injections was stained with anti-mouse TNF-␣ Ab. Although there was extensive TNF-␣ production and deposition deciduae from C5ϩ/ϩ mice treated with aPL-IgG (b), minimal TNF-␣ was present in the absence of C5 activation due to either C5 deﬁciency (c) or treatment with anti-C5 mAb (1 mg, Downloaded from i.p.) (d).

failure. Indeed, we noted that the appearance of TNF-␣ in the sublytic C5b-9 (41). Placental tissues exposed to hypoxia-reoxy- http://www.jimmunol.org/ decidua and in the circulation was coincident with complement genation in vitro (the stimulus associated with activation of com- activation within the inflamed decidua and on the extraembryonic plement (42)) express and secrete increased levels of TNF-␣, un- membranes (13). Both observations were made as early as 30 min derscoring the possibility that trophoblasts themselves are a source after aPL-IgG injection, indicating that TNF-␣ may be an early of proinflammatory cytokines (43). indicator of embryonic or fetal damage. In summary, our findings demonstrate that TNF-␣ is a critical The deleterious effects of TNF-␣ on pregnancy may be direct mediator of aPL Ab-induced injury, and that it is released in re- and indirect. TNFRs expressed on the trophoblast modulate cell sponse to complement activation. Our results fit into a larger proliferation and differentiation in normal pregnancy (10, 31). Ex- schema consistent with evidence from murine models of myocar- pression of TNFRI is constitutive, whereas expression of TNFRII ditis, sepsis, and rheumatoid arthritis, and suggest that tissue dam- by guest on September 24, 2021 is developmentally programmed (32). In vitro studies have shown age induced as a result of complement activation is mediated by that TNF-␣ induces apoptosis of cytotrophoblasts, suggesting that production of TNF-␣ (42, 44). However, because blockade of aberrant expression of TNF-␣ may have harmful effects on pla- TNF-␣ does not completely protect pregnancies, it is likely that cental development and function (33–35). However, regulated other effector pathways contribute to fetal demise. The variability TNF-␣ expression in the developing placenta may be essential at in the effectiveness of TNF-␣ inhibition that we observed is in specific stages of pregnancy, because PEG sTNFRI-induced block- keeping with the heterogeneous responses to TNF-␣ blockade seen ade of TNF-␣ in mice treated with NH-IgG was associated with a in murine models of Ab-induced arthritis (45) and in patients with modest increase in pregnancy failure. Finally, deficient hormone rheumatoid arthritis. Thus, inhibitors of TNF-␣ may provide ef- synthesis by the corpus luteum as a consequence of increased fective treatment for some patients with APS and recurrent preg- TNF-␣ may also interfere with pregnancy survival (36–39). nancy loss. In conclusion, therapies directed at TNF-␣, already in TNF-␣ production may induce abortion indirectly by activating use to treat other inflammatory diseases (46–48), merit more in- endothelial cells and leukocytes. Our previous work showing that tense scrutiny in pregnant women with APS, but the therapeutic mice lacking ICAM-1 are protected from aPL Ab-associated preg- window for TNF-␣ blockade in pregnancy needs to be carefully nancy complications emphasizes the importance of endothelial ac- defined. tivation in fetal injury (40). TNF-␣-stimulated neutrophils and monocytes release inflammatory mediators, including reactive ox- idants, proteolytic enzymes, and complement components, that di- Acknowledgments rectly damage decidual tissue, accelerate alternative pathway ac- We are grateful to our colleagues for generously providing reagents, tivation, enhancing C5 cleavage, and thereby trigger release of Dr. Ulrich Feige (Amgen) for PEG sTNFRI, Dr. Michael Holers (Univer- sity of Colorado Health Sciences Center, Denver, CO) for anti-C5 mAb, more TNF-␣, which further amplifies local inflammation. In addi- and Dr. Paolo Casali (University of California, Irvine, CA) for human aPL tion, stimulation of effector cells by cytokines alters their response mAb. We thank Marta Guerra for help in preparing the manuscript. to complement activation products, and a major effect of C3a and C5a is recruitment of leukocytes that release such cytokines. It is also possible that trophoblasts amplify local damage by secreting References TNF-␣ themselves, in response to complement split products or 1. Wilson, W. A., A. E. Gharavi, T. Koike, M. D. Lockshin, D. W. Branch, J. C. Piette, R. Brey, R. Derksen, E. N. Harris, G. R. Hughes, et al. 1999. Inter- assembly of the terminal membrane attack complex of comple- national consensus statement on preliminary classification criteria for definite ment, C5b-9, on the cell membrane. Indeed, in an experimental antiphospholipid syndrome: report of an international workshop. 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