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Prenatal exposure to maternal infection alters cytokine expression in the placenta, amniotic fluid, and fetal brain
Schizophrenia Research 47 (2001) 27–36 www.elsevier.com/locate/schres
Prenatal exposure to maternal infection alters cytokine expression in the placenta, amniotic fluid, and fetal brain Ari Urakubo a, L. Fredrik Jarskog a,d, Jeffrey A. Lieberman a,d, John H. Gilmore a,d, * a Department of Psychiatry, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA b Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA c Department of Radiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA d UNC Mental Health and Neuroscience Research Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA Received 3 September 1999; accepted 24 January 2000
Abstract Prenatal exposure to infection appears to increase the risk of schizophrenia and other neurodevelopmental disorders. We have hypothesized that cytokines, generated in response to maternal infection, play a key mechanistic role in this association. E16 timed pregnancy rats were injected i.p. with Escherichia coli lipopolysaccharide (LPS) to model prenatal exposure to infection. Placenta, amniotic fluid and fetal brains were collected 2 and 8 h after LPS exposure. There was a significant treatment effect of low-dose (0.5 mg/kg) LPS on placenta cytokine levels, with significant increases of interleukin (IL)-1b (P<0.0001), IL-6 (P<0.0001), and tumor necrosis factor-a (TNF-a) (P= 0.0001) over the 2 and 8 h time course. In amniotic fluid, there was a significant effect of treatment on IL-6 levels (P=0.0006). Two hours after maternal administration of high-dose (2.5 mg/kg) LPS, there were significant elevations of placenta IL-6 (P<0.0001), TNF-a (P<0.0001), a significant increase of TNF-a in amniotic fluid (P=0.008), and a small but significant decrease in TNF-a (P=0.035) in fetal brain. Maternal exposure to infection alters proinflammatory cytokine levels in the fetal environment, which may have a significant impact on the developing brain. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Interleukin-1b; Interleukin-6; Neurodevelopmental disorders; Schizophrenia; Tumor necrosis factor-a
1. Introduction A variety of epidemiological evidence indicates that prenatal exposure to infection increases the risk of schizophrenia. An excess of late winter/early spring births has consistently been found in * Corresponding author. Present address: Department of Psychiatry, CB# 7160, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA. Tel.: +1-919-966-6971; fax: +1-919-966-7659. E-mail address: [email protected] (J.H. Gilmore)
patients with schizophrenia; this excess has been attributed to infectious, nutritional, or other environmental factors ( Torrey et al., 1997; Mortensen et al., 1999). There is a significant association between obstetrical complications, broadly defined, and the ultimate development of schizophrenia (Jones et al., 1998; Dalman et al., 1999; Hultman et al., 1999; for review see Geddes and Lawrie, 1995); many of these studies include infection during pregnancy as an obstetrical complication.
0920-9964/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0 9 2 0 -9 9 6 4 ( 0 0 ) 0 0 03 2 - 3
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Most research on prenatal exposure to infection and schizophrenia has focused on a specific viral infection — that of influenza. The weight of the evidence indicates that maternal influenza infection during pregnancy is associated with a higher incidence of schizophrenia in offspring (reviewed in McGrath and Murry, 1995). Other studies found that a variety of maternal infections during pregnancy were associated with an increased incidence of schizophrenia in offspring, including bronchopneumonia, other respiratory infections, and varicella zoster, ( Watson et al., 1984; Torrey et al., 1988; O’Callaghan et al., 1994; Brown et al., 2000). Thus it likely that the association between in utero or early postnatal exposure to infection and schizophrenia is a more general phenomenon related to infection, and not limited to a single etiologic viral agent such as influenza. The pathological mechanisms underlying prenatal and perinatal risk factors for schizophrenia, including infection, remain largely unstudied. We have hypothesized that pro-inflammatory cytokines generated by the immune system are important mediators of the association between maternal infection, abnormal brain development, and increased risk for schizophrenia and other neurodevelopmental disorders (Gilmore and Jarskog, 1997). Cytokines are known to regulate normal brain development and have been implicated in abnormal brain development (Merrill, 1992; Mehler et al., 1995; Mehler and Kessler, 1997). Pro-inflammatory cytokines are neurotoxic to a variety of developing neurons in vitro. For example, we found that interleukin (IL)-1b, IL-6, and tumor necrosis factor-a ( TNF-a) decrease survival of fetal dopaminergic and serotonergic neurons in vitro (Jarskog et al., 1997). IL-1b decreases neuron survival in primary cultures of embryonic rat hippocampus (Arajujo and Cotman, 1995) and TNF-a potentiates glutamate excitotoxicity in cultures of fetal cortical neurons (Chao and Hu, 1994). Peripheral administration of Escherichia coli lipopolysaccharide (LPS ) is a well-characterized model of infection in rodents, and has been found to stimulate cytokine expression in the central nervous system (CNS ) of adult rodents (Quan et al., 1999; Pitossi et al., 1997; Gatti and Bartfai,
1993; Meyer et al., 1997). LPS administered to pregnant mice increases IL-1a, IL-6, and TNF-a in the maternal circulation and IL-1a and IL-6 in amniotic fluid (Fidel et al., 1994). Maternal LPS administration in rats and hamsters produces a variety of CNS abnormalities, including enlarged ventricles, microcephaly, and neuronal necrosis (Ornoy and Altschuler, 1976; Collins et al., 1994). LPS does not enter the fetal circulation (Goto et al., 1994), thus any effect on fetal brain development would be a result of the maternal, placental, or fetal immune system response to LPS or other systemic response to infection. This animal model was utilized to characterize the response of proinflammatory cytokines in the placenta, amniotic fluid, and fetal brain to maternal infection to begin to determine the role that these cytokines play in the association between maternal infection, abnormal brain development, and increased risk for schizophrenia and other neurodevelopmental disorders.
2. Methods Two separate experiments were conducted. In the initial experiment, gestational day 16 timed pregnancy Sprague–Dawley rats (Zivic-Miller, Allison Park, PN ) were injected with 0.5 mg/kg LPS i.p. (E. coli, O55: B5; Sigma, St. Louis, MO) and sacrificed by decapitation 2 or 8 h later after a brief exposure to ether. The uterine horns containing the embryonic day 16 fetuses were surgically removed. Amniotic fluid was aspirated with a syringe; placenta and whole fetal brain were dissected. All samples were immediately frozen on dry ice and stored at −80°C. In the second study, gestational day 16 timed pregnancy Sprague– Dawley rats received 2.5 mg/kg of LPS or saline i.p. and were sacrificed after 2 h. N=6 for each control and treatment group. Three separate litters were used for each control and treatment group in experiment 1. In experiment 2, two separate litters were used for each control and treatment group. The fetal brain and placental tissue (100– 200 mg) was placed in 10–30 volumes of 50 mM Tris–HCl buffer (pH 7.4) with 0.6 M NaCl, 0.2%
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Triton X-100, 1% BSA, 1 mM benzamidine, 0.1 mM benzethonium chloride, and 0.1 mM PMSF. Samples were homogenized (PowerGen 125, Fisher Scientific, Pittsburgh, PA) on ice for 30 s and sonicated (Sonic Dismembrator 60, Fisher Scientific, Pittsburgh, PA) for 10 s at 10 mV. Samples were centrifuged at 12 000 rpm for 20 min and the supernatants were aliquoted and frozen at −80°C until assays were performed. IL-1b, IL-6, and TNF-a protein levels were determined using a two-site enzyme linked immunosorbent assay ( ELISA) for recombinant rat IL-1b, IL-6 and TNF-a according to the manufacturer’s directions (Biosource, Camarillo, CA). Briefly, samples were added to a 96-well microplate coated with a primary antibody (Ab) followed by a biotinylated secondary Ab to rat IL-1b (coating: mouse monoclonal anti-rat IL-1b Ab, detection: rabbit polyclonal anti-rat IL-1b Ab), IL-6 (coating and detection: sheep polyclonal anti-rat IL-6 Ab), and TNF-a (coating and detection: rabbit polyclonal anti-mouse TNF-a). A streptavidin–peroxidase-labeled Ab was added, followed by the chromogen tetramethyl benzidine/horseradish peroxidase. The reaction was stopped and the optical density was measured at 450 nm using a microplate reader ( Vmax, Molecular Devices, Sunnyvale, CA). The limits of detection for IL-1b, IL-6, and TNF-a were 3 pg/ml, 8 pg/ml, and 4 pg/ml, respectively. The mean intra-assay coefficients of variance for IL-1b, IL-6, and TNF-a were 7.0%, 8.3%, and 13.8%, respectively. Samples and standards were run in triplicate. LPS and control samples were run in the same assay on the same plate. Statistical analyses were performed using Prism 3.0 (GraphPad Software, San Diego, CA). For experiment 1, a two-way ANOVA was used to determine effect of both treatment and time. For experiment 2, differences in cytokine levels were compared with an unpaired t-test. Significance was set at 0.05 using a two-tailed test for all analyses.
3. Results There was a significant treatment effect of lowdose (0.5 mg/kg) LPS on placenta cytokine levels, with significant increases of IL-1b (P<0.0001),
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IL-6, (P<0.0001), and TNF-a (P=0.0001) over the 2 and 8 h time course (see Fig. 1). In amniotic fluid, there was a significant effect of treatment on IL-6 levels (P=0.0006); there was no effect on TNF-a levels, while IL-1b levels were undetectable (see Fig. 1). There was no significant effect of treatment observed in the fetal brain for any of the cytokines at this lower LPS dose (see Fig. 1). A significant time effect was observed in TNF-a levels in the fetal brain (P=0.011). Two hours after maternal administration of high-dose (2.5 mg/kg) LPS, there were significant elevations of placenta IL-6 (P<0.0001), TNF-a (P<0.0001) and a trend for an increase in IL-1b (P=0.06, see Fig. 2). There was a significant increase of TNF-a in amniotic fluid (P=0.008), while levels of IL-1b and IL-6 were undetectable in either condition (see Fig. 2). In the fetal brain there was a small but significant decrease in TNF-a (P=0.035), while there was no significant change in IL-6 or IL-1b (see Fig. 2).
4. Discussion In this animal model, maternal exposure to LPS acutely increases IL-6 and TNF-a levels in the amniotic fluid and acutely increases IL-1b, IL-6, and TNF-a in the placenta. High-dose LPS decreases TNF-a in the fetal brain. Given the mounting evidence that cytokines play important roles in the development of neurons and glial cells, changes in levels of these pro-inflammatory cytokines in the fetal environment may contribute to abnormal brain development associated with prenatal exposure to infection. These findings offer support for the hypothesis that pro-inflammatory cytokines play a key mechanistic role in the association between maternal infection and increased risk for schizophrenia and other neurodevelopmental disorders. Maternal exposure to high-dose LPS decreased TNF-a in the fetal brain, indicating that maternal infection can have a direct impact on the cytokine levels in the developing fetal brain. The contribution of blood cytokines to that measured in brain is minimal (Meyer et al., 1997; Nguyen et al., 1998). The decrease in fetal brain TNF-a is unexpected
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Fig. 1. Cytokine levels in amniotic fluid, placenta, and fetal brain at 2 and 8 h after maternal injection of 0.5 mg/kg LPS. Assay dilutions for IL-1b: brain 1:60, placenta 1:20, amniotic fluid 1:2; for IL-6 and TNF-a: brain 1:30, placenta 1:10, amniotic fluid 1:1 (neat).
(especially in the setting of increased placental and amniotic fluid TNF-a), as TNF-a levels are typically increased after LPS exposure in adult rats (Meyer et al., 1997; Nguyen et al., 1998; Nadeau and Rivest, 1999). This result suggests that the CNS
cytokine response to infection is developmentally regulated and may be different in fetal compared to adult brain; alternatively, additional factors in the fetal environment may underlie this differential response to LPS exposure. The mechanism that
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Fig. 2. Cytokine levels in amniotic fluid, placenta, and fetal brain at 2 h after maternal injection of 2.5 mg/kg LPS. Dilutions for all assays were: brain 1:30, placenta 1:10, amniotic fluid 1:1 (neat), except IL-6 brain which was 1:20.
leads to decreased TNF-a in fetal brain is unclear and may represent a down-regulation of TNF-a expression or an increased uptake and binding of TNF-a to its cellular receptors, preventing detection. Finally, it is interesting to note that TNF-a
levels in fetal brain have a diurnal variation, similar to that described in mature rat brain (Floyd and Krueger, 1997; Bredow et al., 1997). Maternal exposure to LPS caused an increase of IL-1b, IL-6, and TNF-a in the placenta. A time
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course of individual cytokine responses to LPS is suggested, with IL-6 and TNF-a peaking at 2 h after injection, whereas IL-1b became more elevated at 8 h. This pattern differs from known cytokine cascades in plasma in which LPS administration induces initial high levels of TNF-a followed successively by peaks of IL-1 and IL-6 (Abbas et al., 1994), though is similar to that described in mouse placenta after inoculation of the uterus with E. coli (Hirsch et al., 1999). In the amniotic fluid, high-dose LPS caused an increase of TNF-a at 2 h, while low-dose LPS caused an increase of IL-6, especially at 8 h. In the high-dose experiment, IL-6 levels were undetectable in both the saline and LPS conditions. This likely reflects small differences in the baseline levels of IL-6 which are close to the limit of detection for this assay. It is possible that highdose LPS may induce additional modifiers, such as corticosterone, which would decrease cytokine expression (Goujon et al., 1996), though the fact that high-dose LPS increases placental IL-6 argues against this possibility. Increased IL-6 after LPS correlates with clinical data which indicate that amniotic fluid IL-6 levels are a sensitive marker of infection (Greig et al., 1993; Yoon et al., 1995; Greci et al., 1998). In addition, elevated IL-6 in amniotic fluid is found to be correlated with chorioamnionitis, even in the absence of positive cultures from the amniotic fluid (Andrews et al., 1995). Elevated IL-6 levels at diagnostic amniocentesis predict fetal demise; this is hypothesized to reflect subclinical intrauterine infection ( Wenstrom et al., 1996). The mechanism by which the maternal and placental immune systems interact with the fetus and its immune system is not well characterized, although several studies have begun to elucidate this relationship. Maternally generated cytokines have been reported to cross the placenta and regulate cell growth and development in the fetus, as granulocyte colony stimulating factor (G-CSF ) injected into pregnant rats can stimulate fetal granulopoiesis (Medlock et al., 1993). Maternal transforming growth factor-b1 ( TGF-b1) can cross the placenta and ‘rescue’ TGF-b1 null embryos (Letterio et al., 1994). Low amounts of IL-1b, IL-6, and TNF-a have been found to cross
intact amnion, chorion, and decidua in culture ( Kent et al., 1994). In contrast, Carbo et al. (1998) reported that TNF-a administered to uninfected pregnant rats does not cross the placenta, and suggest that cytokines that are found within the fetus are produced in its organs. Cytokines may be more likely to cross the blood–placenta barrier in the setting of infection, as pro-inflammatory cytokines can disrupt the blood–brain barrier (Quagliarello et al., 1991; de Vries et al., 1996). Studies indicate that maternally administered LPS does not enter the fetal circulation (Goto et al., 1994) and that intraperitoneal injection of LPS does not increase permeability of the blood–brain barrier (Bickel et al., 1998). However, LPS can disrupt the blood–brain barrier if administered topically to the cerebral microcirculation (Mayhan, 1998) or intracisternally ( Wispelwey et al., 1988), and it is possible that high doses of LPS may cross the placenta into the fetal circulation. The placenta is known to generate cytokines in response to infection; human amniochorionic membranes produce IL-1b, IL-6, and TNF-a in response to LPS in vitro ( Fortunato et al., 1996). In the setting of labor, TNF-a is derived from placental macrophages, whereas IL-1b and IL-6 are released from placental endothelial cells (Steinborn et al., 1998). It is not known whether these cell types are the source of cytokines in the setting of infection. In chorioamnionitis, serum levels of IL-1b and IL-6 in the human fetus were associated with the histologic severity of infection but not maternal serum levels of these cytokines, indicating that the source of these cytokines in the fetal circulation was the placenta (Salafia et al., 1997). The precise mechanisms by which cytokines generated in response to maternal infection enter the fetal brain remain to be elucidated. It is hypothesized that maternally generated cytokines cross the placenta and enter the fetal circulation. In addition, cytokines generated by the placenta in response to exposure to infectious agents, LPS, or maternal cytokines are also likely to gain entry into the fetal circulation. More recently, a fetal inflammatory response (especially of IL-6) to maternal infection has been demonstrated that is
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associated with severe neonatal morbidity, including CNS morbidity (Gomez et al., 1998); therefore, cytokines generated by the fetus may also play an important role in the relationship between maternal infection and altered brain development. Pro-inflammatory cytokines have emerged as critical factors in the etiology of cerebral palsy. They are likely mediators of the association between maternal intrauterine infection and the development of white matter damage (Adinolfi, 1993; Leviton, 1993; Dammann and Leviton, 1997, 1998; Nelson et al., 1998). Elevated levels of IL-6 in fetal cord blood, and of IL-6 and IL-1b in amniotic fluid are associated with white matter lesions in premature infants ( Yoon et al., 1996, 1997a). IL-6 and TNF-a are highly expressed in astrocytes and microglial cells in periventricular leukomalacia (Deguchi et al., 1996; Yoon et al., 1997b). White matter damage and cerebral palsy as outcome of prenatal exposure to infection and prematurity offer evidence of how more subtle changes in the developing white and/or gray matter that are associated with schizophrenia may occur in the setting of infection. There is increasing evidence of subtle white matter abnormalities in patients with schizophrenia (Buchanan et al., 1998; Lim et al., 1999) and we have recently reported a case of early onset schizophrenia in a female with premature birth and periventricular leukomalacia on neonatal ultrasound (Gilmore et al., 1999). Alternatively, prenatal exposure to increased proinflammatory cytokine levels could also alter neuron programmed cell death, differentiation or ‘connectivity’, mechanisms implicated in schizophrenia. In adult rodents, peripheral infection activates cytokine expression in the choroid plexus and ependyma (Nadeau and Rivest, 1999; Quan et al., 1999); if a similar activation occurs in the developing brain, developing neurons and glia in areas adjacent to the ventricles, including the germinal matrix, would be especially vulnerable (Sarnat, 1992, 1995). Finally, we have recently found that amniotic fluid levels of nerve growth factor and brain derived neurotrophic factor are decreased in human pregnancies complicated by infection (Marx et al., 1999). There is evidence of cytokine regulation of neurotrophic factor expression in glia (Hesse et al., 1998); altered neuro-
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trophic factor expression may be a secondary mechanism by which maternal infection could alter neuron development. Much of the prenatal cortical development that has been implicated in schizophrenia occurs after birth in rats. However, alterations in pro-inflammatory cytokines in the maternal–fetal unit documented in this study are consistent with clinical studies in humans and indicate that maternal infections in humans can significantly increase proinflammatory cytokines in the fetal brain environment. The ultimate impact of the cytokine response to maternal infection on fetal brain development is probably dependent both on the timing and severity of the infection in relation to critical periods of brain development, as well as on interactions with environmental and genetic risk factors. There is increasing evidence that prenatal infection is associated with premature birth (Greci et al., 1998; Gibbs et al., 1992; Arntzen et al., 1998), fetal growth restriction (Beckmann et al., 1993), the signs of birth asphyxia such as low APGAR scores and neonatal seizures (Grether and Nelson, 1997), as well as pre-eclampsia (Hsu and Witter, 1995). Therefore many of the obstetrical complications associated with increased risk of schizophrenia, including low birth weight and short gestation (Jones et al., 1998), pre-eclampsia (Dalman et al., 1999; Kendell et al., 1996), and perinatal hypoxia and low APGAR scores (Dalman et al., 1999), may in fact be associated with clinical or sub-clinical infection. The presence of chorioamnionitis has largely been ignored as a risk factor for schizophrenia, though is a major cause of perinatal complications. In summary, this study provides evidence that maternal infection can significantly change levels of pro-inflammatory cytokines in the prenatal brain environment, and supports the hypothesis that pro-inflammatory cytokines play an important role in neurodevelopmental disorders associated with prenatal exposure to infection, including schizophrenia. Further studies that elucidate the nature of the cytokine response to prenatal infection and its impact on brain development are required.
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Acknowledgements This work was supported by the Stanley Foundation (J.H.G.), The Holderness Foundation (A.U.), MH00537 (J.A.L.), and NIMH Center Grant MH33127 (J.A.L.).
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Johnston, K.E., Hemstreet, G.P., 1996. Elevated amniotic fluid interleukin-6 levels at genetic amniocentesis predict subsequent pregnancy loss. Am. J. Obstet. Gynecol. 175, 830–833. Wispelwey, B., Lesse, A.J., Hansen, E.J., Scheld, W.M., 1988. Haemophilus influenzae lipopolysaccharide-induced blood– brain-barrier permeability during experimental meningitis in the rat. J. Clin. Invest. 82, 1339–1346. Yoon, B.H., Jun, J.K., Romero, R., Park, K.L., Gomez, R., Choi, J., Kim, I., 1997a. Amniotic fluid inflammatory cytokines (interleukin-6, interleukin-1b, and tumor necrosis factor-a), neonatal brain white matter lesions and cerebral palsy. Am. J. Obstet. Gynecol. 177, 19–26. Yoon, B.H., Romero, R., Kim, C.J., Jun, J.K., Gomez, R., Choi, J.H., Syn, H.C., 1995. Amniotic fluid interleukin-6: a sensitive test for antenatal diagnosis of acute inflammatory lesions of preterm placenta and prediction of perinatal morbidity. Am. J. Obstet. Gynecol. 172, 960–970. Yoon, B.H., Romero, R., Kim, C.J., Koo, J.N., Choe, G., Syn, H.C., Chi, J.G., 1997b. High expression of tumor necrosis factor-a and interleukin-6 in periventricular leukomalacia. Am. J. Obstet. Gynecol. 77, 406–411. Yoon, B.H., Romero, R., Yang, S.H., Jun, J.K., Kim, I., Choi, J., Syn, H.C., 1996. Interleukin-6 concentrations in umbilical cord plasma are elevated in neonates with white matter lesions associated with periventricular leuokmalacia. Am. J. Obstet. Gynecol. 174, 1433–1440.
Prenatal exposure to maternal infection alters cytokine expression in the placenta, amniotic fluid, and fetal brain Ari Urakubo a, L. Fredrik Jarskog a,d, Jeffrey A. Lieberman a,d, John H. Gilmore a,d, * a Department of Psychiatry, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA b Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA c Department of Radiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA d UNC Mental Health and Neuroscience Research Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA Received 3 September 1999; accepted 24 January 2000
Abstract Prenatal exposure to infection appears to increase the risk of schizophrenia and other neurodevelopmental disorders. We have hypothesized that cytokines, generated in response to maternal infection, play a key mechanistic role in this association. E16 timed pregnancy rats were injected i.p. with Escherichia coli lipopolysaccharide (LPS) to model prenatal exposure to infection. Placenta, amniotic fluid and fetal brains were collected 2 and 8 h after LPS exposure. There was a significant treatment effect of low-dose (0.5 mg/kg) LPS on placenta cytokine levels, with significant increases of interleukin (IL)-1b (P<0.0001), IL-6 (P<0.0001), and tumor necrosis factor-a (TNF-a) (P= 0.0001) over the 2 and 8 h time course. In amniotic fluid, there was a significant effect of treatment on IL-6 levels (P=0.0006). Two hours after maternal administration of high-dose (2.5 mg/kg) LPS, there were significant elevations of placenta IL-6 (P<0.0001), TNF-a (P<0.0001), a significant increase of TNF-a in amniotic fluid (P=0.008), and a small but significant decrease in TNF-a (P=0.035) in fetal brain. Maternal exposure to infection alters proinflammatory cytokine levels in the fetal environment, which may have a significant impact on the developing brain. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Interleukin-1b; Interleukin-6; Neurodevelopmental disorders; Schizophrenia; Tumor necrosis factor-a
1. Introduction A variety of epidemiological evidence indicates that prenatal exposure to infection increases the risk of schizophrenia. An excess of late winter/early spring births has consistently been found in * Corresponding author. Present address: Department of Psychiatry, CB# 7160, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7160, USA. Tel.: +1-919-966-6971; fax: +1-919-966-7659. E-mail address: [email protected] (J.H. Gilmore)
patients with schizophrenia; this excess has been attributed to infectious, nutritional, or other environmental factors ( Torrey et al., 1997; Mortensen et al., 1999). There is a significant association between obstetrical complications, broadly defined, and the ultimate development of schizophrenia (Jones et al., 1998; Dalman et al., 1999; Hultman et al., 1999; for review see Geddes and Lawrie, 1995); many of these studies include infection during pregnancy as an obstetrical complication.
0920-9964/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0 9 2 0 -9 9 6 4 ( 0 0 ) 0 0 03 2 - 3
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Most research on prenatal exposure to infection and schizophrenia has focused on a specific viral infection — that of influenza. The weight of the evidence indicates that maternal influenza infection during pregnancy is associated with a higher incidence of schizophrenia in offspring (reviewed in McGrath and Murry, 1995). Other studies found that a variety of maternal infections during pregnancy were associated with an increased incidence of schizophrenia in offspring, including bronchopneumonia, other respiratory infections, and varicella zoster, ( Watson et al., 1984; Torrey et al., 1988; O’Callaghan et al., 1994; Brown et al., 2000). Thus it likely that the association between in utero or early postnatal exposure to infection and schizophrenia is a more general phenomenon related to infection, and not limited to a single etiologic viral agent such as influenza. The pathological mechanisms underlying prenatal and perinatal risk factors for schizophrenia, including infection, remain largely unstudied. We have hypothesized that pro-inflammatory cytokines generated by the immune system are important mediators of the association between maternal infection, abnormal brain development, and increased risk for schizophrenia and other neurodevelopmental disorders (Gilmore and Jarskog, 1997). Cytokines are known to regulate normal brain development and have been implicated in abnormal brain development (Merrill, 1992; Mehler et al., 1995; Mehler and Kessler, 1997). Pro-inflammatory cytokines are neurotoxic to a variety of developing neurons in vitro. For example, we found that interleukin (IL)-1b, IL-6, and tumor necrosis factor-a ( TNF-a) decrease survival of fetal dopaminergic and serotonergic neurons in vitro (Jarskog et al., 1997). IL-1b decreases neuron survival in primary cultures of embryonic rat hippocampus (Arajujo and Cotman, 1995) and TNF-a potentiates glutamate excitotoxicity in cultures of fetal cortical neurons (Chao and Hu, 1994). Peripheral administration of Escherichia coli lipopolysaccharide (LPS ) is a well-characterized model of infection in rodents, and has been found to stimulate cytokine expression in the central nervous system (CNS ) of adult rodents (Quan et al., 1999; Pitossi et al., 1997; Gatti and Bartfai,
1993; Meyer et al., 1997). LPS administered to pregnant mice increases IL-1a, IL-6, and TNF-a in the maternal circulation and IL-1a and IL-6 in amniotic fluid (Fidel et al., 1994). Maternal LPS administration in rats and hamsters produces a variety of CNS abnormalities, including enlarged ventricles, microcephaly, and neuronal necrosis (Ornoy and Altschuler, 1976; Collins et al., 1994). LPS does not enter the fetal circulation (Goto et al., 1994), thus any effect on fetal brain development would be a result of the maternal, placental, or fetal immune system response to LPS or other systemic response to infection. This animal model was utilized to characterize the response of proinflammatory cytokines in the placenta, amniotic fluid, and fetal brain to maternal infection to begin to determine the role that these cytokines play in the association between maternal infection, abnormal brain development, and increased risk for schizophrenia and other neurodevelopmental disorders.
2. Methods Two separate experiments were conducted. In the initial experiment, gestational day 16 timed pregnancy Sprague–Dawley rats (Zivic-Miller, Allison Park, PN ) were injected with 0.5 mg/kg LPS i.p. (E. coli, O55: B5; Sigma, St. Louis, MO) and sacrificed by decapitation 2 or 8 h later after a brief exposure to ether. The uterine horns containing the embryonic day 16 fetuses were surgically removed. Amniotic fluid was aspirated with a syringe; placenta and whole fetal brain were dissected. All samples were immediately frozen on dry ice and stored at −80°C. In the second study, gestational day 16 timed pregnancy Sprague– Dawley rats received 2.5 mg/kg of LPS or saline i.p. and were sacrificed after 2 h. N=6 for each control and treatment group. Three separate litters were used for each control and treatment group in experiment 1. In experiment 2, two separate litters were used for each control and treatment group. The fetal brain and placental tissue (100– 200 mg) was placed in 10–30 volumes of 50 mM Tris–HCl buffer (pH 7.4) with 0.6 M NaCl, 0.2%
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Triton X-100, 1% BSA, 1 mM benzamidine, 0.1 mM benzethonium chloride, and 0.1 mM PMSF. Samples were homogenized (PowerGen 125, Fisher Scientific, Pittsburgh, PA) on ice for 30 s and sonicated (Sonic Dismembrator 60, Fisher Scientific, Pittsburgh, PA) for 10 s at 10 mV. Samples were centrifuged at 12 000 rpm for 20 min and the supernatants were aliquoted and frozen at −80°C until assays were performed. IL-1b, IL-6, and TNF-a protein levels were determined using a two-site enzyme linked immunosorbent assay ( ELISA) for recombinant rat IL-1b, IL-6 and TNF-a according to the manufacturer’s directions (Biosource, Camarillo, CA). Briefly, samples were added to a 96-well microplate coated with a primary antibody (Ab) followed by a biotinylated secondary Ab to rat IL-1b (coating: mouse monoclonal anti-rat IL-1b Ab, detection: rabbit polyclonal anti-rat IL-1b Ab), IL-6 (coating and detection: sheep polyclonal anti-rat IL-6 Ab), and TNF-a (coating and detection: rabbit polyclonal anti-mouse TNF-a). A streptavidin–peroxidase-labeled Ab was added, followed by the chromogen tetramethyl benzidine/horseradish peroxidase. The reaction was stopped and the optical density was measured at 450 nm using a microplate reader ( Vmax, Molecular Devices, Sunnyvale, CA). The limits of detection for IL-1b, IL-6, and TNF-a were 3 pg/ml, 8 pg/ml, and 4 pg/ml, respectively. The mean intra-assay coefficients of variance for IL-1b, IL-6, and TNF-a were 7.0%, 8.3%, and 13.8%, respectively. Samples and standards were run in triplicate. LPS and control samples were run in the same assay on the same plate. Statistical analyses were performed using Prism 3.0 (GraphPad Software, San Diego, CA). For experiment 1, a two-way ANOVA was used to determine effect of both treatment and time. For experiment 2, differences in cytokine levels were compared with an unpaired t-test. Significance was set at 0.05 using a two-tailed test for all analyses.
3. Results There was a significant treatment effect of lowdose (0.5 mg/kg) LPS on placenta cytokine levels, with significant increases of IL-1b (P<0.0001),
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IL-6, (P<0.0001), and TNF-a (P=0.0001) over the 2 and 8 h time course (see Fig. 1). In amniotic fluid, there was a significant effect of treatment on IL-6 levels (P=0.0006); there was no effect on TNF-a levels, while IL-1b levels were undetectable (see Fig. 1). There was no significant effect of treatment observed in the fetal brain for any of the cytokines at this lower LPS dose (see Fig. 1). A significant time effect was observed in TNF-a levels in the fetal brain (P=0.011). Two hours after maternal administration of high-dose (2.5 mg/kg) LPS, there were significant elevations of placenta IL-6 (P<0.0001), TNF-a (P<0.0001) and a trend for an increase in IL-1b (P=0.06, see Fig. 2). There was a significant increase of TNF-a in amniotic fluid (P=0.008), while levels of IL-1b and IL-6 were undetectable in either condition (see Fig. 2). In the fetal brain there was a small but significant decrease in TNF-a (P=0.035), while there was no significant change in IL-6 or IL-1b (see Fig. 2).
4. Discussion In this animal model, maternal exposure to LPS acutely increases IL-6 and TNF-a levels in the amniotic fluid and acutely increases IL-1b, IL-6, and TNF-a in the placenta. High-dose LPS decreases TNF-a in the fetal brain. Given the mounting evidence that cytokines play important roles in the development of neurons and glial cells, changes in levels of these pro-inflammatory cytokines in the fetal environment may contribute to abnormal brain development associated with prenatal exposure to infection. These findings offer support for the hypothesis that pro-inflammatory cytokines play a key mechanistic role in the association between maternal infection and increased risk for schizophrenia and other neurodevelopmental disorders. Maternal exposure to high-dose LPS decreased TNF-a in the fetal brain, indicating that maternal infection can have a direct impact on the cytokine levels in the developing fetal brain. The contribution of blood cytokines to that measured in brain is minimal (Meyer et al., 1997; Nguyen et al., 1998). The decrease in fetal brain TNF-a is unexpected
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Fig. 1. Cytokine levels in amniotic fluid, placenta, and fetal brain at 2 and 8 h after maternal injection of 0.5 mg/kg LPS. Assay dilutions for IL-1b: brain 1:60, placenta 1:20, amniotic fluid 1:2; for IL-6 and TNF-a: brain 1:30, placenta 1:10, amniotic fluid 1:1 (neat).
(especially in the setting of increased placental and amniotic fluid TNF-a), as TNF-a levels are typically increased after LPS exposure in adult rats (Meyer et al., 1997; Nguyen et al., 1998; Nadeau and Rivest, 1999). This result suggests that the CNS
cytokine response to infection is developmentally regulated and may be different in fetal compared to adult brain; alternatively, additional factors in the fetal environment may underlie this differential response to LPS exposure. The mechanism that
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Fig. 2. Cytokine levels in amniotic fluid, placenta, and fetal brain at 2 h after maternal injection of 2.5 mg/kg LPS. Dilutions for all assays were: brain 1:30, placenta 1:10, amniotic fluid 1:1 (neat), except IL-6 brain which was 1:20.
leads to decreased TNF-a in fetal brain is unclear and may represent a down-regulation of TNF-a expression or an increased uptake and binding of TNF-a to its cellular receptors, preventing detection. Finally, it is interesting to note that TNF-a
levels in fetal brain have a diurnal variation, similar to that described in mature rat brain (Floyd and Krueger, 1997; Bredow et al., 1997). Maternal exposure to LPS caused an increase of IL-1b, IL-6, and TNF-a in the placenta. A time
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course of individual cytokine responses to LPS is suggested, with IL-6 and TNF-a peaking at 2 h after injection, whereas IL-1b became more elevated at 8 h. This pattern differs from known cytokine cascades in plasma in which LPS administration induces initial high levels of TNF-a followed successively by peaks of IL-1 and IL-6 (Abbas et al., 1994), though is similar to that described in mouse placenta after inoculation of the uterus with E. coli (Hirsch et al., 1999). In the amniotic fluid, high-dose LPS caused an increase of TNF-a at 2 h, while low-dose LPS caused an increase of IL-6, especially at 8 h. In the high-dose experiment, IL-6 levels were undetectable in both the saline and LPS conditions. This likely reflects small differences in the baseline levels of IL-6 which are close to the limit of detection for this assay. It is possible that highdose LPS may induce additional modifiers, such as corticosterone, which would decrease cytokine expression (Goujon et al., 1996), though the fact that high-dose LPS increases placental IL-6 argues against this possibility. Increased IL-6 after LPS correlates with clinical data which indicate that amniotic fluid IL-6 levels are a sensitive marker of infection (Greig et al., 1993; Yoon et al., 1995; Greci et al., 1998). In addition, elevated IL-6 in amniotic fluid is found to be correlated with chorioamnionitis, even in the absence of positive cultures from the amniotic fluid (Andrews et al., 1995). Elevated IL-6 levels at diagnostic amniocentesis predict fetal demise; this is hypothesized to reflect subclinical intrauterine infection ( Wenstrom et al., 1996). The mechanism by which the maternal and placental immune systems interact with the fetus and its immune system is not well characterized, although several studies have begun to elucidate this relationship. Maternally generated cytokines have been reported to cross the placenta and regulate cell growth and development in the fetus, as granulocyte colony stimulating factor (G-CSF ) injected into pregnant rats can stimulate fetal granulopoiesis (Medlock et al., 1993). Maternal transforming growth factor-b1 ( TGF-b1) can cross the placenta and ‘rescue’ TGF-b1 null embryos (Letterio et al., 1994). Low amounts of IL-1b, IL-6, and TNF-a have been found to cross
intact amnion, chorion, and decidua in culture ( Kent et al., 1994). In contrast, Carbo et al. (1998) reported that TNF-a administered to uninfected pregnant rats does not cross the placenta, and suggest that cytokines that are found within the fetus are produced in its organs. Cytokines may be more likely to cross the blood–placenta barrier in the setting of infection, as pro-inflammatory cytokines can disrupt the blood–brain barrier (Quagliarello et al., 1991; de Vries et al., 1996). Studies indicate that maternally administered LPS does not enter the fetal circulation (Goto et al., 1994) and that intraperitoneal injection of LPS does not increase permeability of the blood–brain barrier (Bickel et al., 1998). However, LPS can disrupt the blood–brain barrier if administered topically to the cerebral microcirculation (Mayhan, 1998) or intracisternally ( Wispelwey et al., 1988), and it is possible that high doses of LPS may cross the placenta into the fetal circulation. The placenta is known to generate cytokines in response to infection; human amniochorionic membranes produce IL-1b, IL-6, and TNF-a in response to LPS in vitro ( Fortunato et al., 1996). In the setting of labor, TNF-a is derived from placental macrophages, whereas IL-1b and IL-6 are released from placental endothelial cells (Steinborn et al., 1998). It is not known whether these cell types are the source of cytokines in the setting of infection. In chorioamnionitis, serum levels of IL-1b and IL-6 in the human fetus were associated with the histologic severity of infection but not maternal serum levels of these cytokines, indicating that the source of these cytokines in the fetal circulation was the placenta (Salafia et al., 1997). The precise mechanisms by which cytokines generated in response to maternal infection enter the fetal brain remain to be elucidated. It is hypothesized that maternally generated cytokines cross the placenta and enter the fetal circulation. In addition, cytokines generated by the placenta in response to exposure to infectious agents, LPS, or maternal cytokines are also likely to gain entry into the fetal circulation. More recently, a fetal inflammatory response (especially of IL-6) to maternal infection has been demonstrated that is
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associated with severe neonatal morbidity, including CNS morbidity (Gomez et al., 1998); therefore, cytokines generated by the fetus may also play an important role in the relationship between maternal infection and altered brain development. Pro-inflammatory cytokines have emerged as critical factors in the etiology of cerebral palsy. They are likely mediators of the association between maternal intrauterine infection and the development of white matter damage (Adinolfi, 1993; Leviton, 1993; Dammann and Leviton, 1997, 1998; Nelson et al., 1998). Elevated levels of IL-6 in fetal cord blood, and of IL-6 and IL-1b in amniotic fluid are associated with white matter lesions in premature infants ( Yoon et al., 1996, 1997a). IL-6 and TNF-a are highly expressed in astrocytes and microglial cells in periventricular leukomalacia (Deguchi et al., 1996; Yoon et al., 1997b). White matter damage and cerebral palsy as outcome of prenatal exposure to infection and prematurity offer evidence of how more subtle changes in the developing white and/or gray matter that are associated with schizophrenia may occur in the setting of infection. There is increasing evidence of subtle white matter abnormalities in patients with schizophrenia (Buchanan et al., 1998; Lim et al., 1999) and we have recently reported a case of early onset schizophrenia in a female with premature birth and periventricular leukomalacia on neonatal ultrasound (Gilmore et al., 1999). Alternatively, prenatal exposure to increased proinflammatory cytokine levels could also alter neuron programmed cell death, differentiation or ‘connectivity’, mechanisms implicated in schizophrenia. In adult rodents, peripheral infection activates cytokine expression in the choroid plexus and ependyma (Nadeau and Rivest, 1999; Quan et al., 1999); if a similar activation occurs in the developing brain, developing neurons and glia in areas adjacent to the ventricles, including the germinal matrix, would be especially vulnerable (Sarnat, 1992, 1995). Finally, we have recently found that amniotic fluid levels of nerve growth factor and brain derived neurotrophic factor are decreased in human pregnancies complicated by infection (Marx et al., 1999). There is evidence of cytokine regulation of neurotrophic factor expression in glia (Hesse et al., 1998); altered neuro-
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trophic factor expression may be a secondary mechanism by which maternal infection could alter neuron development. Much of the prenatal cortical development that has been implicated in schizophrenia occurs after birth in rats. However, alterations in pro-inflammatory cytokines in the maternal–fetal unit documented in this study are consistent with clinical studies in humans and indicate that maternal infections in humans can significantly increase proinflammatory cytokines in the fetal brain environment. The ultimate impact of the cytokine response to maternal infection on fetal brain development is probably dependent both on the timing and severity of the infection in relation to critical periods of brain development, as well as on interactions with environmental and genetic risk factors. There is increasing evidence that prenatal infection is associated with premature birth (Greci et al., 1998; Gibbs et al., 1992; Arntzen et al., 1998), fetal growth restriction (Beckmann et al., 1993), the signs of birth asphyxia such as low APGAR scores and neonatal seizures (Grether and Nelson, 1997), as well as pre-eclampsia (Hsu and Witter, 1995). Therefore many of the obstetrical complications associated with increased risk of schizophrenia, including low birth weight and short gestation (Jones et al., 1998), pre-eclampsia (Dalman et al., 1999; Kendell et al., 1996), and perinatal hypoxia and low APGAR scores (Dalman et al., 1999), may in fact be associated with clinical or sub-clinical infection. The presence of chorioamnionitis has largely been ignored as a risk factor for schizophrenia, though is a major cause of perinatal complications. In summary, this study provides evidence that maternal infection can significantly change levels of pro-inflammatory cytokines in the prenatal brain environment, and supports the hypothesis that pro-inflammatory cytokines play an important role in neurodevelopmental disorders associated with prenatal exposure to infection, including schizophrenia. Further studies that elucidate the nature of the cytokine response to prenatal infection and its impact on brain development are required.
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Acknowledgements This work was supported by the Stanley Foundation (J.H.G.), The Holderness Foundation (A.U.), MH00537 (J.A.L.), and NIMH Center Grant MH33127 (J.A.L.).
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