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FTY720 reduces post-ischemic brain lymphocyte influx but does not improve outcome in permanent murine cerebral ischemia.

Liesz A, Sun L, Zhou W, Schwarting S, Mracsko E, Zorn M, Bauer H, Sommer C, Veltkamp R - PLoS ONE (2011)

Bottom Line: Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment.Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO.This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University Heidelberg, Heidelberg, Germany. Arthur.Liesz@med.uni-heidelberg.de

ABSTRACT

Background: The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms.

Methodology/principal findings: FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls.

Conclusions/significance: In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

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FTY720 decreases cerebral lymphocyte invasion.(A) Brain sections were stained 5d after MCAO for T cells (CD3), B cells (B220), granulocytes (MPO) and activated microglia/macrophages (IBA1). (B) Analysis of absolute cell counts of T cells, B cells, granulocytes and microglia/macrophages per total hemisphere in PBS and FTY720 treated animals at 5d after MCAO (n = 6–10 per group, 2–3 individual experiments).
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pone-0021312-g006: FTY720 decreases cerebral lymphocyte invasion.(A) Brain sections were stained 5d after MCAO for T cells (CD3), B cells (B220), granulocytes (MPO) and activated microglia/macrophages (IBA1). (B) Analysis of absolute cell counts of T cells, B cells, granulocytes and microglia/macrophages per total hemisphere in PBS and FTY720 treated animals at 5d after MCAO (n = 6–10 per group, 2–3 individual experiments).

Mentions: We performed histological analysis for the invasion of systemic leukocytes and for activation of resident microglial cells to test the effect of FTY720 on cellular inflammation after experimental brain ischemia (Fig. 6). We stained histological sections at 5d after MCAO in FTY720 treated mice (1 mg/kg, daily treatment, starting 48 h prior to MCAO) and control animals for the expression of CD3 (T cells), B220 (B cells), MPO (granulocytes) and IBA1 (microglia/macrophages) (Fig. 6A). FTY720 treatment significantly reduced cerebral invasion of T cells and B cells compared to control animals (Fig. 6B). However, MPO-positive cells (i.e. mainly neutrophil granulocytes) did not significantly differ between treatment groups. Individual animals in the FTY720 treated group had substantially increased MPO+ cell counts (Fig. 6B). No significant difference was detectable for the expression of IBA+ cells in brain sections of FTY720 or PBS treated animals, a marker for activated microglia/macrophages [33]. We additionally performed flow cytometric analysis of immune cell subpopulations isolated from brains of PBS- or FTY720-treated animals 5d after MCAO to confirm histological data (Table 2) by a previously reported FACS gating strategy [34]. Correspondingly, we observed a substantial reduction of brain invading T-helper (CD3+/CD4+) and T-effector (CD3+/CD8+) cells in FTY720 treated animals compared to controls. Absolute cell counts per ischemic hemisphere were not significantly different for Granulocytes (CD45+/Gr-1+), NK cells (CD45+/NK1.1+) and for antigen-presenting cells (CD11b+/MHC-II+) between the treatment groups (Table 2).


FTY720 reduces post-ischemic brain lymphocyte influx but does not improve outcome in permanent murine cerebral ischemia.

Liesz A, Sun L, Zhou W, Schwarting S, Mracsko E, Zorn M, Bauer H, Sommer C, Veltkamp R - PLoS ONE (2011)

FTY720 decreases cerebral lymphocyte invasion.(A) Brain sections were stained 5d after MCAO for T cells (CD3), B cells (B220), granulocytes (MPO) and activated microglia/macrophages (IBA1). (B) Analysis of absolute cell counts of T cells, B cells, granulocytes and microglia/macrophages per total hemisphere in PBS and FTY720 treated animals at 5d after MCAO (n = 6–10 per group, 2–3 individual experiments).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3119049&req=5

pone-0021312-g006: FTY720 decreases cerebral lymphocyte invasion.(A) Brain sections were stained 5d after MCAO for T cells (CD3), B cells (B220), granulocytes (MPO) and activated microglia/macrophages (IBA1). (B) Analysis of absolute cell counts of T cells, B cells, granulocytes and microglia/macrophages per total hemisphere in PBS and FTY720 treated animals at 5d after MCAO (n = 6–10 per group, 2–3 individual experiments).
Mentions: We performed histological analysis for the invasion of systemic leukocytes and for activation of resident microglial cells to test the effect of FTY720 on cellular inflammation after experimental brain ischemia (Fig. 6). We stained histological sections at 5d after MCAO in FTY720 treated mice (1 mg/kg, daily treatment, starting 48 h prior to MCAO) and control animals for the expression of CD3 (T cells), B220 (B cells), MPO (granulocytes) and IBA1 (microglia/macrophages) (Fig. 6A). FTY720 treatment significantly reduced cerebral invasion of T cells and B cells compared to control animals (Fig. 6B). However, MPO-positive cells (i.e. mainly neutrophil granulocytes) did not significantly differ between treatment groups. Individual animals in the FTY720 treated group had substantially increased MPO+ cell counts (Fig. 6B). No significant difference was detectable for the expression of IBA+ cells in brain sections of FTY720 or PBS treated animals, a marker for activated microglia/macrophages [33]. We additionally performed flow cytometric analysis of immune cell subpopulations isolated from brains of PBS- or FTY720-treated animals 5d after MCAO to confirm histological data (Table 2) by a previously reported FACS gating strategy [34]. Correspondingly, we observed a substantial reduction of brain invading T-helper (CD3+/CD4+) and T-effector (CD3+/CD8+) cells in FTY720 treated animals compared to controls. Absolute cell counts per ischemic hemisphere were not significantly different for Granulocytes (CD45+/Gr-1+), NK cells (CD45+/NK1.1+) and for antigen-presenting cells (CD11b+/MHC-II+) between the treatment groups (Table 2).

Bottom Line: Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment.Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO.This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University Heidelberg, Heidelberg, Germany. Arthur.Liesz@med.uni-heidelberg.de

ABSTRACT

Background: The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms.

Methodology/principal findings: FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls.

Conclusions/significance: In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia.

Show MeSH
Related in: MedlinePlus