Fingolimod (FTY720-P) Does Not Stabilize the Blood-Brain Barrier under Inflammatory Conditions in an in Vitro Model

Abstract

Breakdown of the blood-brain barrier (BBB) is an early hallmark of multiple sclerosis (MS), a progressive inflammatory disease of the central nervous system. Cell adhesion in the BBB is modulated by sphingosine-1-phosphate (S1P), a signaling protein, via S1P receptors (S1P₁). Fingolimod phosphate (FTY720-P) a functional S1P₁ antagonist has been shown to improve the relapse rate in relapsing-remitting MS by preventing the egress of lymphocytes from lymph nodes. However, its role in modulating BBB permeability-in particular, on the tight junction proteins occludin, claudin 5 and ZO-1-has not been well elucidated to date. In the present study, FTY720-P did not change the transendothelial electrical resistance in a rat brain microvascular endothelial cell (RBMEC) culture exposed to inflammatory conditions and thus did not decrease endothelial barrier permeability. In contrast, occludin was reduced in RBMEC culture after adding FTY720-P. Additionally, FTY720-P did not alter the amount of endothelial matrix metalloproteinase (MMP)-9 and MMP-2 in RBMEC cultures. Taken together, our observations support the assumption that S1P₁ plays a dual role in vascular permeability, depending on its ligand. Thus, S1P₁ provides a mechanistic basis for FTY720-P-associated disruption of endothelial barriers-such as the blood-retinal barrier-which might result in macular edema.

Keywords: FTY720-P; blood-brain barrier; inflammation; rat brain microvascular endothelial cell culture; tight junctions.

Figure 1

Histological characterization of rat brain endothelial cells. Phase contrast image of confluent RBMEC revealed that the cells express a spindle shaped morphology. Micrographs of immunofluorescence staining against Hoechst (blue), CD31, vWF, claudin-5, and occludin (green).

Figure 1

Histological characterization of rat brain endothelial cells. Phase contrast image of confluent RBMEC revealed that the cells express a spindle shaped morphology. Micrographs of immunofluorescence staining against Hoechst (blue), CD31, vWF, claudin-5, and occludin (green).

Figure 2

Transendothelial electrical resistance (TEER) of rat brain microvascular endothelial cell (RBMEC) cultures exposed to inflammatory conditions. (A) Time- and dose-dependent effect of FTY720-P (FTY-P; 1, 10 and 100 nM; n = 4) on TEER in RBMECs exposed to interferon γ and tumor necrosis factor α (I + T; 100 IU each) for 18 h compared with TEER of RBMECs in an inflammatory milieu alone (Vehicle I + T; n = 4) and under homeostatic conditions (Vehicle; n = 4). Hydrocortisone treatment (HC; 550 nM; n = 3) was used as a positive control; (B) Absolute TEER values of RBMECs 18 h after exposure to I + T (100 IU each) and FTY720-P treatment (n = 3 or 4). *** p < 0.001; ns, not significant.

Figure 3

Analysis of apoptosis in rat brain microvascular endothelial cells (RBMECs) exposed to inflammatory conditions and incubated with FTY720-P. (A,B) Western blot analyses and densitometric quantification of the amount of full-length caspase 3 protein in RBMEC cultures after exposure to interferon γ and tumor necrosis factor α (I + T; 100 IU each) and treatment with FTY720-P (FTY-P; 1, 10 and 100 nM; n = 4) for 18 h compared with untreated cultures under inflammatory conditions (Vehicle I + T; n = 4) as well as with cultures under a homeostatic milieu (Vehicle; n = 4); (C) Staurosporine treatment (Stauro; 1 μM for 2 h) was used as a positive control. Ctrl, control; ns, not significant; Veh, vehicle.

Figure 4

Secretion of matrix metalloproteinase (MMP)-2 (A) and MMP-9 (B) by rat brain microvascular endothelial cells after exposure to interferon γ and tumor necrosis factor α (I + T; 100 IU each) and treatment with FTY720-P (FTY-P; 1, 10 and 100 nM; n = 4) for 18 h compared with untreated cultures under inflammatory conditions (Vehicle I + T; n = 4) as well as with cultures in a homeostatic milieu (Vehicle; n = 4); (C) Representative experiment of zymography bands. ns, not significant; Veh, vehicle.

Figure 4

Secretion of matrix metalloproteinase (MMP)-2 (A) and MMP-9 (B) by rat brain microvascular endothelial cells after exposure to interferon γ and tumor necrosis factor α (I + T; 100 IU each) and treatment with FTY720-P (FTY-P; 1, 10 and 100 nM; n = 4) for 18 h compared with untreated cultures under inflammatory conditions (Vehicle I + T; n = 4) as well as with cultures in a homeostatic milieu (Vehicle; n = 4); (C) Representative experiment of zymography bands. ns, not significant; Veh, vehicle.

Figure 5

Effect of FTY720-P (FTY) on the amount of junctional protein of rat brain microvascular endothelial cell (RBMEC) cultures under inflammatory conditions. Western blot analysis and densitometric quantification of the amount of claudin 5 (A,B); occludin (C,D); and ZO-1 (E,F) proteins in RBMECs after exposure to interferon γ and tumor necrosis factor α (I + T; 100 IU each) and treatment with FTY-P (1, 10 and 100 nM; n = 4) for 18 h compared with untreated cultures under inflammatory conditions (Vehicle I + T; n = 4) as well as with cultures in a homeostatic milieu (Vehicle; n = 4). β-Actin was used as loading control. * p < 0.05; *** p < 0.001; ns, not significant; Veh, vehicle.

Figure 5

Effect of FTY720-P (FTY) on the amount of junctional protein of rat brain microvascular endothelial cell (RBMEC) cultures under inflammatory conditions. Western blot analysis and densitometric quantification of the amount of claudin 5 (A,B); occludin (C,D); and ZO-1 (E,F) proteins in RBMECs after exposure to interferon γ and tumor necrosis factor α (I + T; 100 IU each) and treatment with FTY-P (1, 10 and 100 nM; n = 4) for 18 h compared with untreated cultures under inflammatory conditions (Vehicle I + T; n = 4) as well as with cultures in a homeostatic milieu (Vehicle; n = 4). β-Actin was used as loading control. * p < 0.05; *** p < 0.001; ns, not significant; Veh, vehicle.

Figure 6

(A) Representative Western blot analysis of the amount of occludin and β-actin proteins in RBMECs either exposed to interferon γ and tumor necrosis factor α (I + T; 100 IU each) or cultured in physiological medium (Veh) after incubation with FTY720-P (100 nM) in the presence or absence of a specific S1P₁ or S1P₃-receptor antagonist (αS1P₃, αS1P₁; 10 μM each) for 18 h; (B) Representative Western blot analysis of the amount of S1P₁ and β-actin proteins RBMECs either exposed to interferon γ and tumor necrosis factor α (I + T; 100 IU each) or cultured in physiological medium after incubation with FTY720-P (1, 10, and 100 nM) for 18 h; (C) Representative immunofluorescence staining against Hoechst (blue) and S1P₁ (green) in RBMECs.

Figure 7

Effect of FTY720-P (FTY-P) on erk1/2 signaling in rat brain microvascular endothelial cell (RBMEC) cultures exposed to inflammatory conditions and incubated with FTY720-P. (A,B) Western blot analyses and densitometric quantification of the amount of erk1/2 and perk1/2 proteins in RBMECs treated for 90 min with FTY-P (1, 10, and 100 nM; n = 3) and exposed to interferon γ and tumor necrosis factor α (I + T; 100 IU each) compared with untreated cultures under inflammatory conditions (Vehicle I + T; n = 3) as well as to cultures in homeostatic milieu (Vehicle; n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; Veh, vehicle.