Neutralizing anti-interleukin-1β antibodies modulate fetal blood–brain barrier function after ischemia
Graphical abstract
Introduction
Hypoxic–ischemic brain injury is a major contributor to neurologic abnormalities originating in the perinatal period (Vannucci, 2000, Volpe, 2012) and there is evidence to suggest that the onset of brain injury in neonates can commence before birth (Vannucci, 2000, Volpe, 2012). We have recently demonstrated that impaired blood–brain barrier (BBB) function represents a formerly unappreciated major component of hypoxic–ischemic brain injury in the fetus and that decreases in the expression of tight junction (TJ) proteins are associated with increases in BBB permeability (Chen et al., 2012). Therefore, we postulated that these changes could represent a therapeutic target to attenuate ischemia-related abnormalities in fetal BBB function.
Pro-inflammatory cytokines are important mediators of brain injury that cause or exacerbate brain injury after perinatal insults (Leviton et al., 2010, Leviton et al., 2011). Interleukin-1β (IL-1β) is a key mediator of inflammatory responses and one of the best-characterized early response pro-inflammatory cytokines. We have previously shown increases in the expression of IL-1β in the cerebral cortex of fetal sheep after brain ischemia (Sadowska et al., 2012). In addition, elevated levels of IL-1β in the brain can accentuate injury and result in adverse neurological outcomes after hypoxic–ischemic injury (Cai et al., 2000, Pantoni et al., 1998). Therefore, IL-1β could represent an attractive target to attenuate injury to the fetal BBB.
Recent evidence suggests that anti-IL1β neutralizing antibodies can reduce the biological effects of pro-inflammatory cytokines (Yasothan and Kar, 2008). However, despite been recent advances in the potential therapeutic use of monoclonal antibodies (mAbs) (Breda et al., 2011), effective mAbs have not been developed or approved to treat perinatal brain injury. We have recently generated and purified pharmacological quantities of a specific mouse anti-ovine IL-1β mAb (anti-IL-1β mAb) using previously reported protocols and reagents (Rothel et al., 1997) for use i our in vivo fetal sheep studies. We also confirmed that this neutralizing anti-IL-1β mAb is sensitive and specific for the ovine IL-1β protein and that it effectively neutralizes the biological effects of the ovine IL-1β in vitro (Chen et al., 2013).
The BBB, blood–cerebral spinal fluid (CSF), and CSF–brain barriers are important barriers during brain development (Ek et al., 2012, Stonestreet et al., 1996). The cerebrovascular endothelial cells of the BBB are connected by tight junction proteins, and maintain homeostasis of the central nervous system (CNS) by limiting the entry of substances that could alter neuronal function (Ek et al., 2012). Evidence suggests that BBB damage is an important component of hypoxic–ischemic brain injury in the fetus and neonate (Chen et al., 2012, Inder and Volpe, 2000). The integrity of the TJ seal between adjacent endothelial cells is also critical to CNS homeostasis after hypoxic–ischemic injury (Abbott et al., 2010, Chen et al., 2012). However, limited information is available regarding the effects of hypoxia–ischemia on BBB function and the ability of therapeutic agents to attenuate ischemia-related increases in BBB permeability during the perinatal period (Chen et al., 2012) because most studies have been performed in rodents in which vascular access is not feasible (Fernandez-Lopez et al., 2012, Muramatsu et al., 1997).
The neurodevelopment of the fetal sheep brain has many similarities to that of the human fetal and neonatal brains (Back et al., 2006, Barlow, 1969). The development of the sheep fetus at 127 days of gestation is approximately similar to that of the near term human infant (Back et al., 2006, Gunn et al., 1997). In addition, procedures in the current study such as systemic intravenous infusions of anti-IL-1β mAb and physiological kinetic measures of BBB permeability are not readily achievable in fetuses of smaller animals such as rodents.
Given these considerations, we tested the hypotheses that intravenous infusions of anti-IL-1β mAb result in mAb penetration into the brain parenchyma and CSF, attenuate ischemia-related increases in IL-1β protein and BBB permeability, and modify TJ protein expression after ischemic injury in the brain of the ovine fetus.
Section snippets
Materials and methods
The present study was performed after approval by the Institutional Animal Care and Use Committees of The Alpert Medical School of Brown University and Women & Infants Hospital of Rhode Island and in accordance to the National Institutes of Health Guidelines for the use of experimental animals.
Gestational age, brain, and body weights
Gestational age (127 ± 2, 126 ± 2, and 126 ± 1 days), brain (42.7 ± 9.6, 36.5 ± 9.5, and 45.1 ± 7.5 g), and body weights (3.24 ± 0.8, 2.89 ± 0.4, 2.97 ± 0.6 kg) were not different among the placebo-treated sham (n = 5), placebo-treated ischemic (n = 6) and anti-IL-1β mAb-treated ischemic (n = 7) groups, respectively.
Physiological variables in the fetal sheep
The arterial pH, PO2 and PCO2, heart rate, MABP, lactate, and hematocrit values were within the normal physiologic range for our laboratory (Stonestreet et al., 1993) and did not change during the study
Discussion
The overall objectives of the current study were to examine the ability of systemically infused anti-IL-1β mAb to enter the fetal brain after ischemia and to attenuate ischemia-related increases in BBB permeability in the ovine fetus (Chen et al., 2012). The novel findings of the current study were as follows. First, after exposure to ischemia and intravenous infusions of anti-IL-1β mAb, cerebral cortical anti-IL-1β mAb levels increased. Second, intravenous anti-IL-1β mAb infusions attenuate
Conclusion
The pro-inflammatory cytokine IL-1β contributes to BBB dysfunction after ischemic injury in the fetus. Systemic infusions of anti-IL-1β mAb results in penetration of mAb into the fetal brain and has biological effects that include diminishing the ischemia-related endogenous up regulation of IL-1β protein and increases in BBB permeability in the fetal brain. We speculate that treatment of human neonates with anti-IL-1β mAb is feasible as Canakinumab, a fully humanized anti-IL-1β antibody, is
Acknowledgments
We gratefully acknowledge the gift of the ovine IL-1 β pGEX-2T vector and mouse monoclonal cell lines from which we produced the monoclonal antibodies against ovine IL1β from the Commonwealth Scientific and Industrial Research Organization (CSIRO), Livestock Industries, Victoria, Australia.
Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers 1R01-HD-057100, RI-INBRE Collaborative
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Present address: Department of Psychology, Rhode Island College, 600 Mount Pleasant Ave., Providence, RI 02908, USA.
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Present address: Department of mathematics, Diné College, Tsalle, AZ, 86556, USA.