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L. Almulki, K. Noda, R. Amini, A. Schering, S. Nakao, F. Tayyari, K. Thomas, S. Masli, A. Hafezi-Moghadam; Upregulation of P-Selectin Glycoprotein Ligand-1 During Endotoxin Induced Uveitis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):948.
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We have shown a critical role for p-selectin glycoprotein ligand-1 (PSGL-1) blockade in endotoxin-induced uveitis (EIU). Lipopolysaccharides (LPS) is reported to down-regulate PSGL-1 on leukocytes, however, our recent data suggests the opposite. This motivated us to investigate PSGL-1 regulation and function during EIU.
Uveitis was induced in mice by i.p. injection of 150µg LPS. PSGL-1 function was investigated using our autoperfused micro flow chamber, coated with murine P-selectin (5µg/ml). Flow cytometry was performed on peripheral blood leukocytes (PBL) using PE-conjugated anti-PSGL-1 mAb (2PH-1, 1µg/106 cells) or IgG control. To release rolling leukocytes from the vascular wall, normal and EIU mice were treated with either anti-PSGL-1 neutralizing mAb (4RA10) or rat IgG. PE-conjugated goat-anti-rat Ig was used for secondary staining. PBL counts were performed using a Coulter Counter.
Leukocyte rolling velocity was significantly reduced on immobilized P-selectin 6h post LPS (2.7±0.2µm/s, n=4 vs. 4.1±0.2µm/s in normal mice, n=6, p<0.05). However, 24h after LPS leukocytes rolled at similar velocities (4.0±0.6µm/sec, n=4, p=0.8) as those from control mice. In parallel, PBL counts from EIU mice significantly decreased 6h after LPS injection (1074±218/µl, n=6, p<0.01) but were not significantly different from normal controls (2795±337/µl, n=6) by 24h (2160±336/µl, n=4, p=0.1). Flow cytometry revealed a significant decrease in mean fluorescence intensity (MFI) of PSGL-1 in PBLs 6h after LPS injection (219.2±15.2) compared to controls (453.9±43.1, n=4, p<0.01), however, 24h after LPS, PSGL-1 MFI was not significantly different from normal animals (355±59, n=4, p=0.2). PBLs from mice injected with anti-PSGL-1 mAb and LPS at the same time showed significantly higher PSGL-1 MFI 6h after LPS (484.7±52.9, n=3) than those of control mice (386.7±40.9, n=3), while PBL counts did not differ between these groups (2656±438 and 2439±150/µl, respectively, p=0.6), suggesting that the high PSGL-1 expressing leukocytes are adhering to the vascular endothelium. This may explain the leucopenia found 6h after LPS injection.
These results reveal a previously unrecognized upregulation of PSGL-1 during acute inflammation, which may explain the reduced leukocyte rolling velocity on immobilized P-selectin and in the ocular vasculature. These data further support our notion that PSGL-1 may become a key target in treatment of ocular inflammatory diseases.
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