Systemic inflammatory stimulus potentiates the acute phase and CXC chemokine responses to experimental stroke and exacerbates brain damage via interleukin-1- and neutrophil-dependent mechanisms

Abstract

Systemic inflammatory stimuli, such as infection, increase the risk of stroke and are associated with poorer clinical outcome. The mechanisms underlying the impact of systemic inflammatory stimuli on stroke are not well defined. We investigated the impact of systemic inflammation on experimental stroke and potential mechanisms involved. Focal cerebral ischemia was induced by intraluminal filament occlusion of the middle cerebral artery (MCAo). Brain damage and neurological deficit 24 h after MCAo were exacerbated by systemic lipopolysaccharide (LPS) administration. This exacerbation was critically dependent on interleukin (IL)-1, because coadministration of IL-1 receptor antagonist abolished the effect of LPS on brain damage. Systemic administration of IL-1 increased ischemic damage to a similar extent as LPS and also exacerbated brain edema. IL-1 markedly potentiated circulating levels of the acute phase proteins, serum amyloid A and IL-6, and the neutrophil-selective CXC chemokines, KC and macrophage inflammatory protein-2. Neutrophil mobilization and cortical neutrophil infiltration were aggravated by IL-1 before changes in ischemic damage. Neutropenia abolished the damaging effects of systemic IL-1. These data show for the first time that an acute systemic inflammatory stimulus is detrimental to outcome after experimental stroke and highlight IL-1 as a critical mediator in this paradigm. Our data suggest IL-1-induced potentiation of neutrophil mobilization via CXC chemokine induction is a putative mechanism underlying this effect. Our results may help to explain the poorer outcome in stroke patients presenting with infection and may have implications for neurodegenerative diseases involving neurovascular alterations, such as Alzheimer's disease, in which systemic inflammation can modulate disease progression.

Figure 1.

Systemic LPS exacerbates ischemic brain damage via an IL-1-dependent pathway. LPS was administered systemically, and the extent of ischemic damage and neurological deficit was assessed 24 h after MCAo. A, LPS significantly increased the volume of ischemic damage. Coadministration of IL-1RA attenuated the effect of LPS. **p < 0.01 versus vehicle (veh); ^#p < 0.05 versus LPS, one-way ANOVA followed by Student's t test with Bonferroni's correction. Data are presented as mean ± SEM. B, Representative cresyl violet-stained sections illustrate the distribution of ischemic damage after MCAo in vehicle- or LPS-treated mice. C, LPS worsened neurological outcome and coadministration of IL-1RA reduced the number of mice with a severe neurological deficit (score, 3). *p < 0.05 versus vehicle, Kruskal–Wallis test followed by Dunn's multiple-comparison test. Bars show median. n = 6–7 per group.

Figure 2.

Dose-dependent exacerbation of ischemic brain damage by systemic IL-1β. Recombinant IL-1β was administered systemically at the onset of MCAo, and the extent of ischemic damage and neurological deficit was assessed 24 h after MCAo. A, IL-1β induced a dose-dependent exacerbation of ischemic damage. **p < 0.01 versus vehicle (veh), one-way ANOVA followed by Dunnett's multiple-comparison test. Data are presented as mean ± SEM. B, Neurological deficit was exacerbated in a dose-dependent manner by IL-1β. *p < 0.05 versus vehicle, Kruskal–Wallis test followed by Dunn's multiple-comparison test. Bars show median. C, IL-1β exacerbated damage in cerebral cortex but not in the striatum. **p < 0.01 versus vehicle, Student's t test. Data are presented as mean ± SEM; n = 6 per group except for IL-1β (100 IU)-treated group (n = 3) because of high mortality rate (>60%).

Figure 3.

Systemic IL-1β potentiates brain edema and blood–brain barrier permeability after MCAo. Recombinant IL-1β was administered systemically at the onset of MCAo, and the temporal progression of ischemic damage, brain edema, and BBB disruption were assessed. A, IL-1β significantly exacerbated ischemic damage 24 h after MCAo but not 4 and 8 h after MCAo. B, IL-1β significantly increased the extent of edema 8 and 24 h after MCAo. C, Representative brain sections immunostained for IgG. IL-1β markedly increased the intensity and distribution of IgG immunoreactivity 8 and 24 h after MCAo, indicating potentiation of BBB disruption by IL-1β. *p < 0.05, **p < 0.01 versus vehicle (veh), Student's t test. Data are presented as mean ± SEM. Scale bar, 1 mm. n = 6–8 per group.

Figure 4.

Systemic IL-1β exacerbates the acute-phase response after MCAo. Plasma SAA and IL-6 concentrations were measured by ELISA in mice administered vehicle (veh) or IL-1β systemically at the onset of MCAo. A, IL-1β significantly increased the concentration of plasma SAA 4 h after MCAo. B, IL-1β markedly increased the concentration of plasma IL-6 at all time points after MCAo, although the change was statistically significant only 4 h after MCAo. nd, Below detection limit. *p < 0.05 versus vehicle, Student's t test. Data are presented as mean ± SEM. n = 6–8 per group.

Figure 5.

Systemic IL-1β potentiates neutrophilia and cortical neutrophil infiltration after MCAo. A, The number of circulating PMNs was assessed in mice administered vehicle (veh) or IL-1β systemically at the onset of MCAo. IL-1β significantly increased the number of circulating PMNs 8 h after MCAo. The dotted line indicates pre-MCAo value (vehicle and IL-1β groups pooled). B–D, Accumulation of neutrophils in the brain was assessed by anti-MBS-1 immunohistochemistry. Neutrophils were observed in the cerebral vasculature (B) and brain parenchyma (C). Representative brain sections illustrate neutrophil accumulation in cerebral cortex ipsilateral to MCAo 8 h (D, E) and 24 h (F, G) after MCAo. Marked increases in the number of neutrophils infiltrating into cortical tissue were observed in IL-1β-treated mice (E, G) compared with vehicle-treated mice (D, F). H, IL-1β significantly increased the number of neutrophils accumulating in the cerebral cortex 4, 8, and 24 h after MCAo. *p < 0.05, **p < 0.01 versus vehicle, Student's t test. Data are presented as mean ± SEM. Scale bars: A–D, 100 μm; E–G, 25 μm. n = 6–8 per group.

Figure 6.

Systemic IL-1β potentiates circulating CXC chemokine levels after MCAo. Plasma KC and MIP-2 concentrations were measured by ELISA in mice administered vehicle (veh) or IL-1β systemically at the onset of MCAo. A, IL-1β significantly increased the concentration of plasma KC 4 and 8 h after MCAo, although levels were generally lower than 8 h after MCAo. By 24 h after MCAo, KC concentrations had returned to baseline levels. B, IL-1β also significantly increased the concentration of plasma MIP-2 4 h after MCAo but did not alter levels at any other time point. nd, Below detection limit. *p < 0.05, **p < 0.01 versus vehicle, Student's t test. Data are presented as mean ± SEM. n = 6–8 per group.

Figure 7.

Neutropenia attenuates IL-1β-induced exacerbation of ischemic damage. Neutropenia was induced by anti-PMN antibody treatment before induction of MCAo. Mice were administered vehicle (veh) or IL-1β systemically at the onset of MCAo. A, Neutrophil depletion significantly abolished the IL-1β-induced neutrophilia 24 h after MCAo and also markedly reduced the number of circulating PMNs in control IgG-treated mice. MBS-1 immunohistochemistry shows abundant neutrophil accumulation in the cerebral cortex 24 h after MCAo in response to IL-1β treatment in control IgG-treated mice (B) but not in neutropenic mice (C). D, Quantification of MBS-1 immunohistochemistry demonstrated a significant attenuation of the IL-1β-induced neutrophil infiltrate in PMN-depleted mice. E, IL-1β induced a significant threefold increase in ischemic damage. This increase was significantly attenuated by >70% in neutropenic mice. *p < 0.05, **p < 0.01, Student's t test; ^‡‡p < 0.01 versus IgG plus vehicle, ^#p < 0.05 versus IgG plus IL-1β, one-way ANOVA followed by Student's t test with Bonferroni's correction. Data are presented as mean ± SEM. F, Representative cresyl violet-stained brain sections illustrate a reduction in cortical damage. G, IL-1β significantly exacerbated neurological deficit, and PMN depletion significantly reduced the severity of neurological deficit in IL-1β-challenged mice. *p < 0.05, Kruskal–Wallis test followed by Dunn's multiple-comparison test. Bars show median. Scale bar, 250 μm. n = 6–7 per group.