FTY720 attenuates accumulation of EMAP-II+ and MHC-II+ monocytes in early lesions of rat traumatic brain injury

Abstract

FTY720 (Fingolimod) is a novel type of immunosuppressive agent inhibiting lymphocyte egress from secondary lymphoid tissues thereby causing peripheral lymphopenia. FTY720 can inhibit macrophage infiltration into inflammatory lesions under pathological conditions. FTY720 has been clinically evaluated for prophylaxis of allograft rejection and treatment of multiple sclerosis, showing promising immunosuppressive effects. A robust inflammatory response after traumatic brain injury (TBI) plays an important role in the secondary or delayed injuries of TBI. Here we have investigated by immunohistochemistry in a rat TBI model the effects of FTY720 on early cell accumulation into the inflammatory tissue response and on expression of major histo-compatibility complex class II (MHC-II) and endothelial-monocyte activating polypeptide II (EMAP-II). Accumulation of MHC-II(+) or EMAP-II(+) cells became significant 1 day after injury and continuously increased during the early time periods. Further, double-staining experiments confirmed that the major cellular sources of MHC-II were reactive macrophages, however MHC-II(+) cells only constituted a small subpopulation of reactive macrophages. Immediately after TBI, peripheral administration of FTY720 (1 mg/kg in 1 mL saline, every second day) significantly attenuated the accumulation of MHC-II(+) macrophages from Day 1 to 4 and significantly attenuated the accumulation of EMAP-II(+) macrophages/microglia at Day 4. Our findings show that FTY720 attenuates early accumulation of EMAP-II(+) and MHC-II(+) reactive monocytes following TBI, indicating that FTY720 might be a drug candidate to inhibit brain inflammatory reaction following TBI.

1

MHC-II⁺ leukocyte accumulation in rat brain after TBI: FTY720 reduces the early accumulation of MHC-II⁺ and EMAP-II⁺ cells. (A) Bar graph showing numbers of MHC-II⁺ leukocytes in normal rat brain and accumulation in brain lesions of rats treated with saline or FTY720 (1 mg/kg, i.p. injected immediately after TBI and once every second day after injury). For each section, numbers of MHC-II⁺ cells of every rat brain coronal section were counted in three non-overlapping high-power fields (HPFs, × 400 magnification), which were selected from lesional areas that have a maximum of positive cells. Results were given as arithmetic means of positive cells per HPF and standard errors of means (SEM). Statistical analysis was performed by non-parametric t test (Graph Pad Prism 4.0 software). *P < 0.05 and **P < 0.01 compared with their respective saline controls and ++ P < 0.01 compared with the normal control. (B) Bar graph showing accumulation of EMAP-II⁺ leukocytes in brain lesions of saline or FTY720 (1 mg/kg, i.p. injected immediately after TBI and once per day) treated TBI rats at days 1, 2 and 4 after injury. The numbers of EMAP-II⁺ cells of each rat brain coronal section were counted in 3 HPF in the lesioned areas. Results were given as arithmetic means of positive cells per HPF and standard errors of means (SEM). Statistical analysis was performed by non-parametric t test (Graph Pad Prism 4.0 software). **P < 0.01 compared with their respective saline controls.

2

FTY720 attenuates the accumulation of EMAP-II⁺ and MHC-II⁺ cells in the early lesion of rat brains following experimental TBI. (A) In normal rat brain, only few MHC-II⁺ cells (arrow indicated) were occasionally seen in the subarachnoid space. (B) Cells double-stained with MHC-II⁺ (brown) and ED1+ (blue) were found in the lesioned area at Day 2 after TBI and almost all MHC-II⁺ cells co-expressed ED1 but only a small portion of ED1+ cells co-expressed MHC-II. (C-F) Representative photomicrographs showing that expressions of MHC-II (B, C and D) and EMAP-II (E and F) in brain lesions of TBI rats treated by FTY720 was attenuated as compared to brains of saline treated rats at Day 2 (MHC-II) or Day 4 (EMAP-II) after injury. C and E: saline treated rats; D and F: FTY720 treated rats. Original magnification: A-F× 400.