Multiple injections of anti-mouse β2glycoprotein 1 antibody induce FcRγ-dependent fetal growth restriction (FGR) in mice

Placenta 33 (2012) 540e547

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Multiple injections of anti-mouse b2glycoprotein 1 antibody induce FcRg-dependent fetal growth restriction (FGR) in mice R. Kawaguchi a, *, S. Nunomura b, N. Umehara a, T. Nikaido c, B. Huppertz d, T. Tanaka a, C. Ra b a

Department of Obstetrics and Gynecology, Jikei University School of Medicine, 3-25-8 Minatoku Nishi-shibmbashi, Tokyo 105-8461, Japan Division of Molecular Cell Immunology and Allergology, Nihon University Graduate School of Medical Science, Tokyo 173-8610, Japan c Department of Pathology, Jikei University School of Medicine, Tokyo 105-8461, Japan d Institute of Cell Biology, Histology and Embryology, Center for Molecular Medicine, Medical University of Graz, Graz 8010, Austria b

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 31 March 2012

Objectives: The antiphospholipid syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies (aPLs), is a leading cause for thromboembolic events, repeated miscarriage, fetal loss and is a major risk factor for fetal growth restriction (FGR) and preeclampsia. In human, anti-b2 glycoprotein I (ab2GPI) antibody is one of the aPLs and considered to be a specific and important marker for APS. However, pathophysiological changes induced by ab2GPI antibodies in FGR are largely unknown. Methods: In the present study, we developed a murine FGR model induced by multiple injections of WBCAL-1, a well-characterized mouse ab2GPI monoclonal antibody. Results: Administration of WBCAL-1, but not the isotype control antibody and saline, into pregnant mice specifically decreased the size of fetuses and placentas without affecting the number of delivered pups. Also, a significant increase in urinary albumin and electron microscopic changes, such as splitting layers of basal membranes in the placental labyrinth and rearrangement of pores in glomerular endothelial cells, were observed in WBCAL-1 treated mice. WBCAL-1 injection did not induce any changes in blood pressure and typical parameters of blood thromboembolic symptoms. Furthermore, FcRg deficiency protected the fetuses from ab2GPI antibody-induced injuries. Conclusions: Our present findings suggest that proteinuria is a symptom associated with APS-related FGR with placental and renal tissue injuries, and that FcRg might be a molecular target for prevention of ab2GPI antibody-mediated obstetrical pathologies. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Antiphospholipid antibody syndrome (APS) Fetal growth restriction (FGR) Mouse model Anti-b2 glycoprotein I (ab2GPI) antibody FcRg deficient mice

1. Introduction The antiphospholipid syndrome (APS) is characterized by the persistent presence of circulating antiphospholipid antibodies (aPLs). In 1989, APS became a distinct clinical entity independent of systemic lupus erythematosus (SLE) or other autoimmune diseases [1,2]. Although the numbers of new aPLs are steadily increasing, the following antibodies have been adopted as the main laboratory criteria for APS: lupus-like anticoagulants (LA), anti-cardiolipin (aCL) and anti-b2 glycoprotein I (ab2GPI) [3]. Accordingly, clinical criteria for APS (Sydney criteria) are (i) one or more unexplained fetal deaths at or beyond the 10th week of gestation, (ii) one or more premature births before the 34th week, because of eclampsia or severe preeclampsia or placental insufficiency, or (iii) three or more unexplained consecutive spontaneous abortions prior to the

* Corresponding author. Tel.: þ81 3 3433 1111x3521; fax: þ81 3 3433 1219. E-mail address: [email protected] (R. Kawaguchi). 0143-4004/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2012.03.010

10th week [3]. Recurrent miscarriage occurs in 1% of the general population and more than 20% of women with recurrent miscarriage are positive for circulating aPLs [4]. In fact, aPLs-positive women show lower live-birth rates compared to aPLs-negative women [5]. This is in line with studies demonstrating that pregnancy loss in women with aPLs is highly associated with fetal death, which is not the case in women without aPLs [4,6]. APLs are recognized as a tissue injury factor stimulating the complement cascade, activating platelets and endothelial cells as well as clotting factors [7]. Increase of aPLs has been associated with inadequate blood supply to the growing placenta (“placental insufficiency”) which may lead to spontaneous abortion, fetal growth restriction (FGR), preeclampsia, stillbirth, or placental abruption [5]. FGR is relatively common in the above obstetrical pathologies. Its prevalence is 8% in the general population [8], while about 50% of stillbirths and 25% of preeclampsia cases are associated with FGR [9,10]. Moreover, low birth weight caused by placental insufficiency is associated with the development of the metabolic syndrome later in life [11].

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Previous studies have reported that injection of undefined human aPLs from serum of APS patients induces FGR or fetal resorption in mice [7,12e15]. We also regard aPLs as being responsible for placental insufficiency and FGR. Moreover, there is a need to investigate whether and how specific monoclonal aPL antibodies are linked to APS-related FGR. We used WBCAL-1, a well-characterized mouse ab2GPI monoclonal antibody (MAb), to develop a novel murine FGR model, since (i) among aPLs, aCL and ab2GPI antibodies are thought to be crucial players in human APS, (ii) the ab2GPI antibody is more specific for thrombosis in patients with APS compared to the aCL antibody [16], and (iii) the ab2GPI antibody is more important for clinical diagnosis and therapy compared to the aCL antibody [17,18]. WBCAL-1 was established from NZW  BXSB F1 mice, a model of spontaneous APS [19], has g2a constant regions and binds to cardiolipin only in the presence of b2GPI in vitro [20,21]. Activating Fcg-receptors (FcgR-s) are involved in various pathogenic antibody-mediated responses. Currently, four major subtypes, FcgRI, FcgRII, FcgRIII and FcgRIV, are known in mice. FcRg chain is a common signaling subunit for cell surface expression of these FcRs (except FcgRII) and thereby FcRg deficient mice are recognized as FcgRI/FcgRIII/FcgRIV knockout mice. A recent study employing FcRg deficient mice indicated that activation of FcgRs is dispensable for murine FGR induced by administration of human aPLs [12]. In the present study, we investigated the effect of the ab2GPI antibody WBCAL-1 on fetal growth and maternal wellbeing in pregnant mice. We used this model to assess the consequences of early stages of aPL in pregnant mice. Moreover, we examined the role of activating FcRg in ab2GPI antibody-induced FGR employing FcRg deficient mice. With this targeted and translational approach we could not identify pathological mechanisms but rather open the way for in depth studies on the link between aPL and IUGR.

mice were generated from a C57BL/6J-derived ES cell line, and targeted deletion of the murine FcRg gene was confirmed by genomic PCR [22]. Female mice were mated with previously isolated males, and the presence of a vaginal plug was defined as day 0 of pregnancy. WBCAL-1 was injected into all pregnant mice three times during pregnancy. On days 8, 11, and 14 of pregnancy, WBCAL-1 (500 mg/kg) (CAL group: WT; n ¼ 13, FcRg/ n ¼ 9), IgG2ak (500 mg/kg) (IgG group: WT; n ¼ 13, FcRg/; n ¼ 9), or saline (10 ml/g) (NC group: WT; n ¼ 16, FcRg/ n ¼ 10) was injected into mice via tail vein. Mice were sacrificed on day 17 of pregnancy, blood was collected from the axillary artery while the mouse was in a coma using ether, and at the same time, urine was taken from the bladder. Immediately after death of the mice, uterus, kidney and liver were dissected, fetuses and placentas were weighed. Systolic and diastolic blood pressures were measured before and after mating (day 0 of pregnancy), prior to injections (days 8, 11 and 14 of pregnancy), and prior to sacrifice (day17 of pregnancy). As pilot studies, five mice were treated with WBCAL-1 (1000 mg/kg) on gestational days 8, 11, 14, and four mice were treated with WBCAL-1 (500 mg/kg) on gestational days 5, 11, 14.

2. Materials and methods

2.5. Statistical analyses

2.1. Preparation of antibody

Data are expressed as mean  SD. Statistical analysis was performed using oneway ANOVA and post hoc tests, Tukey, student’s t-test or ManneWhitney U test. P < 0.05 was considered significant.

Mouse ab2GPI MAb (clone WBCAL-1: the hybridoma was a kind gift from Prof. Dr. T Koike of the Hokkaido University, Japan) was prepared in Dr. Ra’s laboratory [20]. Briefly, the hybridoma secreting WBCAL-1 (3  106 cells) were intraperitoneally injected into BALB/c nude mice pretreated with pristine. After WBCAL-1 injection, ascitic fluid was collected and the ab2GPI MAb was purified by a MAbTrap Kit (GE Healthcare). Purity of the MAb was usually over 95% (data not shown). Mouse IgG2ak antibody was purchased from BD Biosciences (MPC-11; San Diego, CA, USA) as an isotype control. 2.2. Murine pregnancy model Following the approval of the ethical committee of Nihon University, all experiments were performed in accordance with the guidelines for the care and use of laboratory animals of Nihon University. Adult mice (12e20 weeks old) were used in all experiments, and were bred in an animal facility under specific pathogen-free conditions. C57BL/6J wild type mice (WT) were purchased from Charles River Laboratories (Kanagawa, Japan) as a littermate control. FcRg-deficient (FcRg/)

2.3. Analysis of physiological parameters EDTA blood was used for the blood cell count and the test of coagulation factors such as APTT (activated partial thromboplastin time), while serum was used to measure GPT (glutamate-pyruvate transaminase), TG (triacylglycerol), BUN (blood urea nitrogen), CRE (creatinine) and UA (uric acid) within 24 h by SRL, Inc (Tokyo, Japan). For the detection of urine protein, the Albuwell M kit (Exocell; PA, USA) was used, an indirect competitive ELISA designed to monitor kidney function in the mouse by measuring urinary albumin. Systolic and diastolic blood pressures were measured by a programmable sphygmomanometer BP-98A (Softron; Tokyo, Japan) using a tail-cuff in a calm setting. Since the measurement takes about 10 s and needs a non-moving tail, three measurements were performed when the mice were entirely composed, and the average value was used for further analysis. 2.4. Microscopic analysis Tissue samples were fixed in formalin and embedded in paraffin. Sections were stained with H&E and PAS for light microscopy. For electron microscopy, 1 mm3 of fresh tissue was fixed with 1.2% glutaraldehyde solution and post-fixed with 1% phosphate-buffered osmium tetroxide. Following dehydration, tissue blocks were embedded in Epon 812 and cut with an ultramicrotome. After staining with uranyl acetate and lead citrate, ultrathin sections were examined in an electron microscope (Hitachi 8000, Japan).

3. Results 3.1. Anti-mouse b2GP1 monoclonal antibody induces fetal growth restriction in an FcRg-dependent manner In two pilot studies we injected (1) 1000 mg/kg of WBCAL-1 at gestational days 8, 11 and 14 or (2) 500 mg/kg of WBCAL-1 at gestational days 5, 11 and 14. In pilot study (1) the high dose of WBCAL-1 was lethal for one out of five mice and in four of out five pregnant mice it lead to hypertension (Table 1). Large numbers of infarctions and thrombosis were confirmed in the placentas obtained from the dead mouse (data not shown). In pilot study (2)

Table 1 Studies on WT maternal mice treated with different applications of WBCAL-1.

Pilot study (1) Pilot study (2) Performance method

Injection schedule

Concentration of WBCAL-1

Number of pregnant mice

Number of mice with fetal resorptions

Number of mice with premature delivery

Number of dead pregnant mice

Number of mice with increased SBP

8, 11, 14 5, 11, 14 8, 11, 14

1000 mg/kg 500 mg/kg 500 mg/kg

5 4 13

0 3 0

0 1 (day 15) 0

1 (day 17) 0 0

4a 0 1

Schedule: gestational days of treatment. Concentration: concentration of the antibody at one treatment. Number of pregnant mice: Pregnancies were confirmed by their vaginal plug on day 0 of gestation. Number of mice with fetal resorptions: These resorptions were confirmed on day 11 of gestation. Number of mice with increased SBP: Mice having more than 140 mmHg systolic blood pressure on day 17 of gestation. a The four mice with hypertension include the mouse died after assessment of blood pressure.

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Fig. 1. WBCAL-1 induces a decrease in fetal and placental weight in wild type pregnant mice Wild type and FcRg/ mice were treated with WBCAL-1 (CAL), isotype antibody (IgG), or saline (NC) intravenously on days 8, 11 and 14 of pregnancy. On day 17, fetuses (A, C) and placentas (B, C) were dissected from pregnant mice. The data shown are the mean  SD. One-way ANOVA and Tukey post hoc test were performed in the CAL and the FcRg/ group (*, p < 0.05; **, p < 0.01). Significant differences were observed between fetal weights of WT-IgG and FcRg/-IgG, and WT-NC and FcRg/-IgG by student t-test (*, p < 0.05). This can be explained by a similarly reduced weight of adult FcRg/ mice compared to WT mice. Representative results are displayed at the bottom.

a first injection of 500 mg/kg of WBCAL-1 at day 5 of pregnancy resulted in miscarriages in three out of four mice and in one premature delivery (Table 1). Therefore, we started the MAb administration with the lower dosage at day 8 of pregnancy. We examined whether antibody administration into WT pregnant mice induced FGR by injecting 500 mg/kg of ab2GPI MAb (WBCAL-1), 500 mg/kg of control IgG2a MAb or saline at days 8, 11, and14 of pregnancy (Table 1). Fig. 1 shows that multiple injections of WBCAL-1 significantly decreased fetal and placental weight in WT mice (Fig. 1A, B: CAL group, black bars), suggesting that the ab2GPI MAb induced FGR in mice. Next, we examined the role of activating FcRg in our model by using FcRg/ mice. Fig. 1 shows that in FcRg/ mice WBCAL-1 failed to induce a decrease in fetal and placental weight (Fig. 1A, B: FcRg/ group, grey bars). These results indicate that FcRg was indispensable for WBCAL-1-mediated FGR. We summarized

pregnancy outcomes in the two groups (Table 2). Interestingly, there were no significant differences in the number of delivered pups between the two groups. 3.2. Anti-mouse b2GP1 monoclonal antibody does not induce alterations in blood parameters The examination of blood parameters showed an association with obstetric complications. However, no significant differences were observed in any of the blood parameters (Table 3). 3.3. Urine albumin increased in WBCAL-1 treated wild type mice without any signs of hypertension Since clinical conditions of FGR and preeclampsia are known to be closely related [23], we measured blood pressure in our FGR model. Fig. 2A shows that blood pressure did not change between

Table 2 Application of WBCAL-1 antibody decreased fetal and placental weight in wild type mice. Mouse type

FcRg/

WT

Group

CAL

IgG

NC

CAL

IgG

Number of pregnant mice Fetal number per litter Fetal weight (mg) Fetal weight (Index)a Placental weight (mg) Index of placental/fetal weight

13 7.1 815 89.0 101 0.12

13 6.2 939 102 109 0.12

16 6.9 916 100 109 0.12

9

9

    

2.1 78* 8.5* 9* 0.1

    

2.2 106 11.6 12 0.1

    

2.0 116 12.7 10 0.1

6.1 849 104 106 0.12

    

1.3 127 15.5 11 0.1

5.8 853 104 113 0.13

NC     

1.9 97 11.8 23 0.2

10 6.3 817 100 115 0.13

    

1.8 113 13.8 19 0.2

Female C57BL/6J wild type (WT) and FcRg deficient (FcRg/) mice were treated with WBCAL-1 (CAL), isotype antibody (IgG), or saline (NC) intraperitoneally on days 8, 11 and 14 of pregnancy. Mice were sacrificed on day 17. After uteri were dissected, the number of fetuses and placentas were counted and weighted. Fetal resorptions were hardly observed. Asterisks (*) denote statistically significant differences in fetal and placental weight between CAL with NC, and CAL with IgG among WT mice (*, p < 0.05: Turkey’s test). No significant differences were observed in fetal and placental weight among FcRg/ mice. a Defined fetal weight of NC as 100 and calculated for each group. Data represent mean  SD.

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(CAL: 13.0  7.2 mg/dl vs. IgG: 4.1  3.9 mg/dl, p < 0.01, vs. NC: 5.9  3.0 mg/dl, p < 0.05) (Fig. 2B). In contrast, the same WBCAL-1 treatment failed to increase levels of urinary albumin in FcRg/ mice (CAL: 5.5  2.0 mg/dl, IgG: 4.7  4.0 mg/dl, NC: 3.7  3.8 mg/dl) (Fig. 2B).

Table 3 Data of blood samples obtained from treated mice on day 17 of pregnancy. FcRg/

Mouse type

WT

Group

CAL

Number of 13 pregnant mice Platelets (10x4/ml) APTT (sec) GPT (IU/ml) TG (mg/dl) BUN (mg/dl) CRE (mg/dl) UA (mg/dl)

50  21 25 47 51 24 0.2 1.4

     

IgG

NC

CAL

IgG

NC

13

16

9

9

10

41  13

41  12

40  13

36  17

36  19

1 26  20 53  58 59  0 22  0 0.3  0.2 2.0 

1 34  27 43  23 31  3 22  0 0.2  0.2 1.7 

19 27  26 43  15 40  4 23  0 0.2  0.8 1.8 

2 24  22 32  15 27  4 22  0 0.3  1.1 2.6 

8 30  17 46  17 34  3 24  0 0.2  1.9 2.9 

543

1 35 16 3 0 2.3

Female C57BL/6J wild type (WT) and FcRg deficient (FcRg/) mice were treated with WBCAL-1 (CAL), isotype antibody (IgG), or saline (NC). Mice were sacrificed on day 17. Results of laboratory tests are expressed as mean  SD. APTT, activated partial thromboplastin time; GPT, glutamate-pyruvate transaminase; TG, triacylglycerol; BUN, blood urea nitrogen; CRE, creatinine; UA, uric acid. Turkey’s test was done for these statistical analyses.

the two groups. Different to the human, where blood pressure is lower during pregnancy (especially in the third trimester), this is not the case in pregnant mice. Here, blood pressure decreases slightly early in pregnancy and stays low until mid-gestation, then starts to rise reaching the level prior to pregnancy. This is the reason why there are no changes in blood pressure between days 8 and 17 in control animals, and especially in the mouse strain used in this study, C57BL/6J [24,25]. On the other hand, WT mice treated with WBCAL-1 exhibited a higher level of urinary albumin compared to control groups

3.4. Using light microscopy no histological changes could be detected in the placental labyrinth and renal glomeruli of mice treated with WBCAL-1 As demonstrated in Fig. 1, placentas in mice injected with WBCAL-1 were apparently small, but their shape and gross morphology were not different compared with those of the control groups. Therefore, we histologically examined the placental labyrinth to reveal putative differences in the placental compartment with the closest interactions between maternal and fetal blood. Although smaller fetuses and placentas were delivered from WT mice treated with WBCAL-1, no significantly different findings were detected compared with controls and FcRg/ mice (Fig. 3AeD). In addition, although WT mice treated with WBCAL-1 showed symptoms of a nephrotic syndrome, a nonspecific disorder caused by renal damage leading to leakage of large amounts of proteins into urine, histological alterations such as focal segmental sclerosis in renal glomeruli were not observed in H&E stained sections (Fig. 3EeG). In the WT-CAL group no remarkable findings were obtained, although tissues were analyzed at high magnification and staining with PAS to visualize membranes (Fig. 3IeL). Also, no significant findings in placentas and kidneys obtained from FcRg/ mice treated with antibodies and saline were obtained (Fig. 3H shows FcRg/-CAL while for IgG and NC data are not shown).

A

B

Fig. 2. WBCAL-1 increases levels of urine albumin in wild type pregnant mice Wild type and FcRg/ mice were treated with WBCAL-1 (CAL), isotype antibody (IgG), or saline (NC) as in Fig. 1. (A) Blood pressure was measured before antibody treatment (day8) and before sacrifice (day17). Solid and dashed lines denote systolic and diastolic blood pressure, respectively. (B) Urine albumin in the absorbed urine liquid before sacrifice on day 17 of pregnancy. In A and B, the data shown are the mean  SD. ManneWhitney U test was performed between day 8 and day 17 (A), and one-way ANOVA and Tukey post hoc test were performed in the CAL and the FcRg/ group (B) (*, p < 0.05; **, p < 0.01).

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Fig. 3. WBCAL-1 induces no major morphologic alterations in wild type pregnant mice Histological data of placental labyrinth (AeD) and kidney glomeruli (EeH) obtained from day 17 of pregnancy. Injection of WBCAL-1 (WT-CAL), IgG2ak (WT-IgG), or saline (WT-NC) into wild type mice, and of WBCAL-1 into knock-out mice (FcRg/ -CAL) on days 8, 11 and 14 days of pregnancy. Higher power fields of placental labyrinth (I, K) and kidney glomeruli (J, L) from WT-CAL treated mice were stained with H&E (I, J) or the PAS method (K, L). Even at higher magnification using light microscopy no remarkable changes compared to controls were observed. Original magnification: AeH; 400, I-L; 800.

3.5. Ultrastructural changes in placental labyrinth and renal glomeruli are detected by electron microscopy We finally examined whether electron microscopy detects ultrastructural changes in placental labyrinth and renal glomeruli. Performing electron microscopy on six independent experiments in each group, splits of blood vessel basal membranes in the labyrinth and rearrangement of pores in endothelial cells of glomeruli were only detected in WT pregnant mice (day17) treated with WBCAL-1 (Fig. 4A, A0 and E). Split layers in the labyrinth were not observed in placentas from IgG and NC groups (Fig. 4C,D). In addition, although rearrangements of pores in endothelial cells of glomeruli were sometimes observed in controls (Fig. 4H), massive rearrangement of pores was only observed in WT mice treated with WBCAL-1 (Fig. 4E). In FcRg/ mice, no effects on placental and renal tissues were observed even after treatment with WBCAL-1 (Fig. 4B, B0 and F). 4. Comment In the present study, smaller fetuses and placentas as well as morphological alterations of the labyrinth basement membrane were induced in mice treated with the ab2GP1 MAb WBCAL-1. An altered placental/fetal weight ratio was reported in severe cases of FGR [11]. The mouse model in this study is considered non severe FGR and/or in an early stage of APS-related FGR, since (i) a two-fold increased dosage of the antibody-induced PIH (Table 1), (ii) early administration of the antibody-induced miscarriage or premature delivery (Table 1), and (iii) the placental/fetal weight (P/F) ratio was not different among the groups (Table 2). We observed an increase in urinary albumin in mice treated with WBCAL-1 although no increase in blood pressure was observed.

Preeclampsia is generally defined as substantial proteinuria and new hypertension after 20 weeks of gestation [26]. An increased relative risk of 2.7e9.7 (95% CI) to develop preeclampsia has been reported in women with aPLs [26,27]. Importantly, several clinical studies reported that significant proteinuria is one of the notable candidates for the predictive clinical manifestation of preeclampsia in patients with FGR. Pregnant women with FGR babies developing preeclampsia later in pregnancy show 10-times more proteinuria prior to the onset of preeclampsia compared to women not developing preeclampsia later in pregnancy [23]. Further, significantly increased proteinuria has been observed in pregnant women who had slightly increased diastolic or systolic blood pressure although defined as being normotensive [10]. Although no significant changes were observed in blood pressure and blood chemistry analyses in mice treated with WBCAL-1, an increased level of urinary albumin was observed. Since all CAL cases had histological changes in the endothelial cell pores of the glomeruli, it may depend on the individual renal function whether the level of proteinuria increased or not. However, our APS-mouse model alerts that APS-related FGR may be associated with pathological changes in various organs resulting in proteinuria and may well be followed by pregnancy induced hypertension. We believe that the data of the present study could suggest to the clinical routine that the first symptom of APS-related FGR and preeclampsia might be the advance of proteinuria rather than blood pressure changes. We are very interested in this aspect and are trying to investigate this aspect in clinical research in the moment. In other studies mice of the SLE nephritis model showed focal segmental necrosis in their kidneys [28e30]; hence, we expected to observe glomerulosclerosis in our APS-FGR mouse model as well. However, a general rather than focal rearrangement of endothelial

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Fig. 4. WBCAL-1 induces micro-pathological changes in wild type pregnant mice Wild type (A, A0 , C, D, E, G, H) and FcRg/ mice (B, B0 and F) were treated with WBCAL-1 (A, A0 , B, B0 , E and F), isotype antibody (C and G), or saline (D and H) as in Fig. 1. Electron micrographs of placental labyrinth (AeD) and renal glomeruli (EeH) obtained from day 17 of pregnancy are shown. Placenta (AeD); SL; Splits of the basal membrane (BM) layer are detected at the direction of arrows, BM; basal membrane, E; endothelial cell, PL; placental labyrinth, RBC; red blood cell. A0 and B0 were taken from broken-line squares in A and B. Kidney (EeH); BP; endothelial basal pores of glomeruli, and rearrangements of BP are shown in dashed line circles, PP; podocyte pedicles, C; glomerular capillary. Results shown are representative of six independent experiments in each group.

cell pores in the glomeruli was observed. The different findings in the two models could be explained by the fact that nephritis in lupus is frequently induced by anti-dsDNA antibodies which bind to glomerular membrane-associated chromatin [31], while ab2GP1 antibodies especially target to the trophoblastic membrane in

placenta and to endothelial cells (ECs) leading to obstetrical pathologies [32,33]. Clearly, in our study we could not identify the mechanisms leading to FGR in the WT mice opposite to the FcRg/ mice. With the set up used in this study a direct binding of the WBCAL-1

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antibody to placental or kidney tissues was not doable. Therefore, we can only speculate on which pathways have been used by the aPLs antibody. It has been reported that the complement system and tissue factor (TF) are involved in mediating fetal injury via aPLs promoted neutrophil activation [7,12,34e36]. Hence, it is tempting to speculate on the pathogenesis induced by WBCAL-1 in our mouse model: Trophoblast: WBCAL-1 may directly bind to b2GP1 on the placental trophoblast [33] and may directly or indirectly lead to damage of the trophoblast basal membrane. The indirect pathway could include TF expressed on neutrophils, which in response to the complement system contributes to the formation of the split layer of the labyrinth’s basal membrane [36]. This in turn could lead to dysregulation of oxygen and nutrient exchange, and subsequently could result in FGR. Glomeruli: Regarding the results in the kidney glomeruli, the microdisturbances could be the result of two mechanisms. (1) Trophoblast could be dysregulated by direct effects of WBCAL-1. This could lead to release of bioactive trophoblast debris in combination with the increase of various components such as inflammatory cytokines into the maternal circulation, which in turn may lead to a maternal disease similar to human preeclampsia [26]. (2) Glomerular damage and microvascular injury could be directly induced by WBCAL-1 antibodies through TF activation and subsequent effects on endothelial cells via complement-dependent and/or independent pathways [37]. In the report cited in the second pathway [37], the authors described ultrastructural glomerular changes and ‘loss of fenestration’ of glomerular endothelial cells, similar to our findings. However, it seems as if the disturbances described in [37] were more severe than those found in our study. Regarding the involvement of the Fc-receptor, the present study clearly indicates that WBCAL-1 elicits FGR and resorption in an FcgR-dependent manner. The findings differ from other murine FGR models where injection of human polyclonal aPLs induces FGR in an FcgR-independent manner [12]. It was previously reported that administration of human polyclonal IgG into mice nonspecifically activates FcgRIIB, the inhibitory murine FcgR [38]. Passive transfer of human IgG prepared from patients with rheumatoid arthritis elicits FcgR-dependent arthritis in FcgRIIB/ mice but not in WT mice. Among the four mouse IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3), mouse IgG2a, the Ig subclass of WBCAL-1, shows low-affinity binding to the FcgRIIB [39]. Based on these findings, we believe that low-affinity binding of IgG2a to the FcgRIIB may explain why WBCAL-1 can induce FcgR-dependent FGR. In addition, we consider that other differences in each experimental setting might be influencing factors of the FcgRmediated response. Our data clearly indicate that both pathophysiological changes were nearly abolished in FcRg/ mice, suggesting that WBCAL-1 antibodies do not have a direct effect on the endothelial pores and the trophoblast basement membrane in our model. The wide expression pattern of the FcR on trophoblast and endothelial cells in the placenta [40] as well as neutrophils and glomerular endothelial cells [22], renders any putative pathomechanism uncertain. With our limited data mechanisms for FcgR-dependent inflammatory pathways in our model mouse remain unclear. However, we speculate that tissue injuries of the placental labyrinth and renal glomeruli may in turn result in FGR and substantial proteinuria in WBCAL-1 treated mice. In APS-related murine models induced by human polyclonal antibodies, the heterologous complement (mouse) system was activated [7,12,35,36]. Actually, in human, increased complement deposition was found on trophoblast cells and platelets in APS patients [41,42]. However, although local complement activation is

significantly increased in most of the experimental settings in murine models, less activation can be detected in histological analysis of human tissues. In APS pregnancies no clear relationship has been reported between pregnancy outcome and therapy [43]. Therefore, we need to be aware that a mouse model does never reflect any human disease in all its aspects. At the same time, murine models have certainly provided us with many important ideas for human diseases, and thus, we believe that our experimental mouse model employing WBCAL-1 is valuable for investigating the role of FcRs in aPLs-induced FGR. We expect that elucidation of FcgR activation induced by aPLs will provide further information for the treatment of aPLs-mediated obstetrical diseases. Authors’s roles KAWAGUCHI; study design, execution, analysis, manuscript drafting, and critical discussion. NUNOMURA; execution, manuscript drafting, and critical discussion. UMEHARA; execution and analysis. NIKAIDO; execution, analysis, and critical discussion. HUPPERTZ; manuscript rewriting and critical discussion. TANAKA; manuscript rewriting and critical discussion. RA; study design and critical discussion. Funding This work was supported by the Grants-in-aid for Scientific Research from the Nihon University (C. Ra), and by the Grants-inaid from the Jikei University (T. Tanaka and T. Nikaido). Acknowledgment We gratefully acknowledge Prof. Dr. Koike for supplying the hybridoma for WBCAL-1. We are grateful to two technicians, Nozomi Watanabe employed by Nihon University and Kazumi Kabumoto employed by Jikei University for their help and expertise. References [1] Harris EN. Syndrome of the black swan. Br J Rheumatol 1987;26:324e6. [2] Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC, et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome: report of an international workshop. Arthritis Rheum 1999;42:1309e11. [3] Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4: 295e306. [4] Oshiro BT, Silver RM, Scott JR, Yu H, Branch DW. Antiphospholipid antibodies and fetal death. Obstet Gynecol 1996;87:489e93. [5] Derksen RH, Khamashta MA, Branch DW. Management of the obstetric antiphospholipid syndrome. Arthritis Rheum 2004;50:1028e39. [6] Salafia CM, Parke AL. Placental pathology in systemic lupus erythematosus and phospholipid antibody syndrome. Rheum Dis Clin North Am 1997;23: 85e97. [7] Holers VM, Girardi G, Mo L, Mao D, Vialpando CG, Salmon JE, et al. Complement C3 activation is required for antiphospholipid antibody-induced fetal loss. J Exp Med 2002;195:211e20. [8] Mandruzzato G, Antsaklis A, Botet F, Chervenak FA, Figueras F, Grunebaum A, et al. Intrauterine growth restriction (IUGR). J Perinat Med 2008;36:277e81. [9] Frøen JF, Gardosi JO, Thurmann A, Francis A, Stray-Pedersen B. Restricted fetal growth in sudden intrauterine unexplained death. Acta Obstet Gynecol Scand 2004;83:801e7. [10] North RA, Taylor RS, Schellenberg JC. Evaluation of a definition of preeclampsia. Br J Obstet Gynaecol 1999;106:767e73. [11] Cetin I, Alvino G. Intrauterine growth restriction: implications for placental metabolism and transport. Placenta 2009;30:77e82.

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