We report the novel finding that IL-1 is a mediator of intraamniotic (IA) LPS–induced lung and systemic inflammation and lung maturation in the preterm fetus. The findings are clinically relevant because the experiments used a model of fetal inflammation induced by administration of LPS in the amniotic cavity, which closely models human chorioamnionitis (28
). The chorioamnionitis-induced fetal inflammation causes injury responses in the lung and the brain in both preterm humans as well as preterm sheep (3
). A biologically important conclusion from the present study is that IL-1 signaling in the amniotic compartment is critical to the pathogenesis of chorioamnionitis-induced inflammation. Fetal inflammatory responses are different from those in the adult, because the preterm fetus does not respond to TNF-α (19
). Therefore, it should not be surprising that whereas IL-1 blockade has variable effects on LPS signaling in the adult, IL-1 blockade significantly decreased LPS-induced inflammation in the fetus in this study.
The biological activity of IL-1 is mediated by two different gene products, IL-1α and IL-1β (21
). These two proteins share only 25% amino acid identity, but signal via binding of either ligand to IL-1 receptor-1 (IL-1R1). The IL-1 receptor antagonist (IL-1ra), a member of the IL-1 cytokine family, binds to IL-1R1 and thus antagonizes biological effects of IL-1. Both IL-1α and IL-1β have been implicated in the pathogenesis of systemic inflammatory disorders that respond to IL-1ra treatment (29
). The fetal tissues that express IL-1β mRNA in response to intraamniotic LPS are the lung and the chorioamnion (16
). Although the predominant site of IL-1β expression in the fetal lung is the inflammatory cells, IL-1α mRNA could not be detected in fetuses exposed to intraamniotic LPS (our unpublished data). We also reported that the LPS receptor Toll-like receptor-4 (TLR4) is expressed in all lung cell types in the fetus and other proteins required for LPS signaling, such as lipopolysaccharide-binding protein, can be detected in the fetal airway (26
). The fetal airway epithelium can also respond to IL-1 because intratracheal injection of IL-1α induces lung inflammation similar to LPS (18
). The present study demonstrated that fetal intraamniotic injection of rhIL-1ra resulted in IL-1 signaling blockade in the amniotic compartment, because little transfer from the amniotic to blood compartment was detected. Taken together, our study suggests that IL-1β secreted by the airway inflammatory cells amplifies LPS-induced lung and systemic inflammation in the fetus. Also, preterm lambs do not have macrophages in the airspaces (33
), suggesting that the primary target of IL-1 receptor blockade is likely to be the airway epithelium. However, the precise mechanisms of how blockade of IL-1 signaling in the amniotic compartment decreases fetal inflammation remain to be identified.
Fetal inflammatory response syndrome has been recognized as a distinct, albeit nebulous entity that is associated with subtle increases in umbilical cord plasma cytokine levels in the absence of bacteremia (5
). By contrast, systemic inflammatory response syndrome in the adult, generally caused by sepsis or trauma, results in a “cytokine storm” causing multiorgan dysfunction and a high risk of death (35
). Despite the subtle increases in plasma cytokines, a systemic inflammatory response induced by chorioamnionitis is postulated to be the proximate cause of fetal organ injury, for example, periventricular leukomalacia (4
), necrotizing enterocolitis (3
), or bronchopulmonary dysplasia (4
). In this study, some of the markers of systemic inflammation (plasma haptoglobin and liver serum amyloid A3 expression) increased 2 days after LPS, whereas other markers (plasma IL-8 and protein carbonyls) increased later, suggesting different mechanisms of induction for these indicators of systemic inflammation. Intraamniotic injection of rhIL-1ra effectively decreased intraamniotic LPS-induced induction of liver serum amyloid, plasma IL-8, protein carbonyls, and blood neutrophil counts. However, rhIL-1ra did not significantly reverse intraamniotic LPS-induced increases in plasma haptoglobin and induction of NOSII expression in the fetal liver (see
Figure E2 in the online supplement). Taken together, intraamniotic inhibition of IL-1 signaling decreased most but not all indicators of chorioamnionitis/LPS-induced fetal systemic inflammatory response. These results demonstrate that, although IL-1 signaling is an important requirement for LPS effects in chorioamnionitis, other pathways downstream of TLR4 signaling also contributes to fetal inflammation.
Lung inflammation is a major consequence of IA LPS injection (28
). IL-1 is an important mediator of IA LPS–induced lung inflammation because rhIL-1ra inhibited IA LPS–induced airway neutrophil and monocyte influx and activation. Transgenic mice expressing IL-1β in the lung epithelium in the perinatal period had pulmonary inflammation and disruption of normal lung architecture (36
). Both LPS and IL-1 receptors share common downstream signaling molecules (37
), and both can induce the expression of IL-6, IL-8, and monocyte chemotactic protein-1 in the perinatal lung (16
). We previously reported that intraamniotic injection of IL-1α and IL-1β (17
), but not TNF-α (19
) or IL-8 (20
), induced robust pulmonary inflammatory cell recruitment, consistent with an important role for IL-1 in chorioamnionitis-induced prenatal inflammation. Because the responses to IL-1α were completely inhibited under our experimental conditions, the present study is informative for inflammatory pathways downstream of TLR4 signaling in the fetus that require IL-1 signaling, in contrast to those that are IL-1 independent. Whereas recruitment and activation of inflammatory cells in the lung were IL-1 dependent, pulmonary expression of the acute-phase reactant serum amyloid A3 was not. Also, intraamniotic rhIL-1ra had dichotomous effects on NOSII and serum amyloid expression in the lung versus the liver, suggesting different mechanisms of induction of these mediators in the lung versus the systemic compartment.
Several studies in adult animals have examined the role of IL-1 signaling in mediating physiological effects of LPS administered both systemically and via the airway. Mice lacking IL-1R1, a major transducer of the IL-1 signal, responded to systemically administered LPS with an acute-phase response indistinguishable from controls (38
). Human IL-1 receptor antagonist modestly decreased systemically administered LPS-induced fever but not plasma IL-6 levels in rats and caused modest reversal of LPS-induced appetite suppression in mice (39
). Treatment with rhIL-1ra did not prevent intraperitoneal LPS-induced preterm labor in mice (41
). Conversely, IL-1 receptor antagonist reduced systemically administered LPS-induced lethality in adult rabbits and mice (42
). IL-1 signaling is required for mediating turpentine-induced inflammation, because mice lacking IL-1R1 were resistant to inflammatory responses induced by subcutaneous turpentine (21
). Similarly, IL-1 signaling is central to the pulmonary inflammation induced by bleomycin (44
). Compared with wild-type mice, mice lacking IL-1R1 had no reduction in airway inflammatory cell influx or airway hyperreactivity induced by LPS given by airway inhalation (45
). On the other hand, rhIL-1ra decreased airway neutrophil recruitment induced by intratracheal LPS by 45% (46
). Collectively, these studies demonstrate that blockade of IL-1 signaling has variable effects on LPS-induced physiological effects in adult animals. In contrast, our results in the preterm fetus demonstrate that IL-1 signaling in the amniotic compartment is a significant mediator and serves to amplify LPS-induced fetal inflammation.
Preterm fetal sheep have low lung surfactant pools and intraamniotic LPS exposure increases surfactant protein mRNAs followed by increased airway surfactant lipid pools, leading to improved lung compliance (12
). In this study, rhIL-1ra decreased intraamniotic LPS-induced pulmonary surfactant protein mRNA, surfactant lipid pool size, and lung compliance. We previously reported that LPS-induced inflammatory cell influx mediated by the integrin CD18 is required to mediate lung maturation (47
). The results from this study demonstrate that IL-1 signaling is an important mediator of IA LPS–induced lung inflammatory cell influx and lung maturation.
Because our studies suggest beneficial effects of rhIL-1ra in chorioamnionitis/LPS-induced lung and systemic inflammation, a valid concern would be the apparently deleterious effects on fetal lung mechanics. In several different chorioamnionitis models, fetal lung inflammation consistently causes lung maturation (16
) and inhibition of inflammatory cell influx in the lung reversed the intraamniotic LPS–induced lung maturation in the fetal sheep (47
). Preterm human fetuses exposed to chorioamnionitis also have clinical lung maturation (2
). Therefore, reversal of the improved lung mechanics may be a necessary consequence of inhibition of inflammation induced by chorioamnionitis. The two clinical strategies to prevent/treat respiratory distress syndrome in clinical use in preterm infants are antenatal maternal glucocorticoids and postnatal surfactants (49
). Effects of the combination of these therapies with rhIL-1ra in chorioamnionitis models remain to be evaluated.