Neutralizing anti-interleukin-1β antibodies modulate fetal blood-brain barrier function after ischemia

Abstract

We have previously shown that increases in blood-brain barrier permeability represent an important component of ischemia-reperfusion related brain injury in the fetus. Pro-inflammatory cytokines could contribute to these abnormalities in blood-brain barrier function. We have generated pharmacological quantities of mouse anti-ovine interleukin-1β monoclonal antibody and shown that this antibody has very high sensitivity and specificity for interleukin-1β protein. This antibody also neutralizes the effects of interleukin-1β protein in vitro. In the current study, we hypothesized that the neutralizing anti-interleukin-1β monoclonal antibody attenuates ischemia-reperfusion related fetal blood-brain barrier dysfunction. Instrumented ovine fetuses at 127 days of gestation were studied after 30 min of carotid occlusion and 24h of reperfusion. Groups were sham operated placebo-control- (n=5), ischemia-placebo- (n=6), ischemia-anti-IL-1β antibody- (n=7), and sham-control antibody- (n=2) treated animals. Systemic infusions of placebo (0.154M NaCl) or anti-interleukin-1β monoclonal antibody (5.1±0.6 mg/kg) were given intravenously to the same sham or ischemic group of fetuses at 15 min and 4h after ischemia. Concentrations of interleukin-1β protein and anti-interleukin-1β monoclonal antibody were measured by ELISA in fetal plasma, cerebrospinal fluid, and parietal cerebral cortex. Blood-brain barrier permeability was quantified using the blood-to-brain transfer constant (Ki) with α-aminoisobutyric acid in multiple brain regions. Interleukin-1β protein was also measured in parietal cerebral cortices and tight junction proteins in multiple brain regions by Western immunoblot. Cerebral cortical interleukin-1β protein increased (P<0.001) after ischemia-reperfusion. After anti-interleukin-1β monoclonal antibody infusions, plasma anti-interleukin-1β monoclonal antibody was elevated (P<0.001), brain anti-interleukin-1β monoclonal antibody levels were higher (P<0.03), and interleukin-1β protein concentrations (P<0.03) and protein expressions (P<0.001) were lower in the monoclonal antibody-treated group than in placebo-treated-ischemia-reperfusion group. Monoclonal antibody infusions attenuated ischemia-reperfusion-related increases in Ki across the brain regions (P<0.04), and Ki showed an inverse linear correlation (r= -0.65, P<0.02) with anti-interleukin-1β monoclonal antibody concentrations in the parietal cortex, but had little effect on tight junction protein expression. We conclude that systemic anti-interleukin-1β monoclonal antibody infusions after ischemia result in brain anti-interleukin-1β antibody uptake, and attenuate ischemia-reperfusion-related interleukin-1β protein up-regulation and increases in blood-brain barrier permeability across brain regions in the fetus. The pro-inflammatory cytokine, interleukin-1β, contributes to impaired blood-brain barrier function after ischemia in the fetus.

Keywords: Blood–brain barrier; Brain; Cytokines; Interleukin-1β; Ischemia–reperfusion; Monoclonal antibody; Ovine fetus; Sheep.

Fig. 1

Changes in ECoG, carotid blood flow and anti-IL-1β mAb concentration during the study. Arrows indicate the timing of the placebo or anti-IL-1β mAb infusions. Non-ischemic placebo-treated sham control (Sham-PL), non-ischemic anti-IL-1β mAb-treated sham control (Sham-mAb), placebo-treated ischemic (Isch-PL), and anti-IL-1β mAb-treated ischemic (Isch-mAb) groups are indicated as open circles connected by dashed lines, open square connected by dashed lines, closed circle connected by solid lines, closed square connected by solid lines, respectively. (A) ECoG decreased during ischemia. ECoG for Sham-PL (n=5), Isch-PL (n=4) and Isch-mAb (n=7) groups plotted against study time in hours on X-axis, before ischemia at baseline, and during ischemia and reperfusion. Isch-PL (n=4) because of inadequate ECoG recordings in two of the fetal sheep in this group. Y-axis is the power spectral densities, PSD-ECoG plotted as the difference from the individually averaged baseline ECoG values. Values are shown as median ± SD. *P<0.05 compared to baseline within Isch-PL and Isch-mAb groups, but not the sham control group. (B) Carotid blood flow plotted as percent change from baseline for the Sham-PL (n=5), Isch-PL (n=6), Isch-mAb (n=7), and Sham-mAb (n=2) groups plotted against study time in hours. Values are shown as mean ± SD. *P<0.001 versus baseline within the ischemic groups (Isch-PL and Isch-mAb). (C) Anti-IL-1β mAb concentrations increase in fetal plasma after the infusions. Concentration (μg/ml) of anti-IL-1β mAb in fetal plasma plotted on the Y-axis against study time in hours on X-axis. The Sham-PL (n=5), Isch-PL (n=6), Isch-mAb (n=7), and the sham-mAb (n=2) groups. The increase in plasma anti-IL-1β mAb concentration was detected within 45 minutes after the onset infusions. *P<0.001 versus baseline values.

Fig. 2

Anti-IL-1β mAb and IL-1β protein concentrations in brain parenchyma and CSF by study group. Scatter grams of anti-IL-1β mAbs within the brain parenchyma (A) and CSF (B) plotted on the Y-axis in ng/g brain tissue and μg/ml, respectively for the Sham-PL (open circles, n=5), Isch-PL (closed circles, n=6), Isch-mAb (closed squares, n=7), and Sham-mAb (open squares, n=2) groups on the X-axis. IL-1β protein concentrations in the brain parenchyma (C) and CSF (D) plotted on the Y-axis in ng/g brain and μg/ml, respectively. Group designations as for A and B, *P<0.05.

Fig. 3

Protein expression of IL-1β, TNF-α, and IL-6 in fetal brain measured by Western-immunoblot. Top panel shows the expression of an internal control standard protein (IC), and Western immunoblots for IL-1β, TNF-α, and IL-6 protein expressions in the Isch-mAb, Sham-PL, and Isch-PL groups. GAPDH is shown as a loading control. The lower panel shows bar graphs for IL-1β, TNF-α, and IL-6 protein expression plotted on the Y-axis as ratios to the IC proteins. *P<0.001 versus Sham-PL group. *P<0.001 versus Isch-PL group.

Fig. 4

Blood-to-brain transfer constants (K_i) in brain regions by study group. Blood-to-brain transfer constants (K_i) measured with AIB plotted on the Y-axis in the Sham-PL (open bars, n=5), Isch-PL (closed bars, n=6), and Isch-mAb (hatched bars, n=7) plotted for brain regions on the X-axis. Sham-PL versus Isch-PL across brain regions (ANOVA: F=7.83, P<0.03); Isch-PL versus Isch-mAb across brain regions (ANOVA: F=5.37, P<0.05). Values are mean ± SD. *P<0.05 vs. sham-PL group, *P<0.05 vs. Isch-PL group.

Fig. 5

K_i values measured with AIB in the cerebral cortex of the Isch-PL (solid circles, n=6), and Isch-mAb (solid squares, n=7) fetuses plotted against the anti-IL-1β mAb concentrations in the cerebral cortex. The least squares linear regression analysis was used to compare the K_i values to the anti-IL-1β mAb concentrations in the cerebral cortex, r = −0.65, n=13, P<0.02.

Fig. 6

Tight junction protein expression in the cerebral cortex, caudate nucleus, cerebellum, and medulla oblongata by study group. Expression of occludin, claudin-1, claudin-5, ZO-1, and ZO-2 proteins in the Sham-PL (open bars, n=5), Isch-PL (closed bars, n=5-6), Isch-mAb (hatched bars, n=7) groups in cerebral cortex (A), caudate nucleus (B), cerebellum (C), and medulla oblongata (D) measured by Western immunoblot. Immunoblots shown above the bar graphs. IC indicates the internal control standard protein derived from the adult cerebral cortex. GAPDH is also shown as a loading control. Values are expressed as mean ± SD. *P<0.05 vs. Sham-PL, *P<0.05 vs. Isch-PL group.

Fig. 7

Schematic representation of the results. In the sham group, the BBB is relatively impermeable (A). Ischemia in the placebo-treated group results in increases in BBB permeability in some brain regions, which is associated with increases in cerebral cortical concentrations of IL-1β protein (B). Systemic anti-IL-1β mAb infusions increase anti-IL-1β mAb levels in the systemic circulation and brain parenchyma, and are associated with decreases in IL-1β and a small increase in TNF-α in the brain parenchyma, and decreases in regional BBB permeability (C).