P-selectin Glycoprotein Ligand-1 Blockade Suppresses Endotoxin Induced Uveitis

Abstract

Purpose:: To investigate the role of P-selectin glycoprotein ligand-1 (PSGL-1) in an experimental model of uveitis.

Methods:: Endotoxin-induced uveitis (EIU) was generated in GFP transgenic and C57Bl/6 WT mice by intraperitoneal injection of 150µg LPS. To investigate functional PSGL-1 upregulation during EIU, we used our recently developed autoperfused micro flow chamber. Briefly, translucent quartz micro chambers (0.4x0.04mm) were coated with 5µg/ml recombinant P-selectin and microsurgically attached via tubing to the carotid artery and the jugular vein of anesthetized mice. Blood flow through the chamber allows live microscopy of leukocyte interaction with the immobilized P-selectin. Leukocyte rolling velocity in the chambers was quantified using normal or EIU animals, 6 and 24h after LPS. To investigate the therapeutic potential of PSGL-1 blockade, the number of leukocytes in the aqueous humor was counted in EIU mice treated with PSGL-1 mAb or isotype control. Retinal leukostasis was quantified using ConA lectin staining. TNF-α and MCP-1 mRNA expression was assessed in the retinal extracts at 6 and 24h after LPS.

Results:: Leukocyte rolling velocity was significantly reduced on immobilized P-selectin at 6h post LPS (2.7±0.2µm/s vs. 4.1±0.2µm/s in normal mice, p<0.05). At 24h after LPS, PSGL-1 blockade reduced leukocyte counts in the aqueous humor of EIU mice by 69.3% (24.7±4.2 and 80.3±24.8 cells/µl, p<0.05), retinal leukostasis by 78.5% (49±5.8 and 227±25.6 leukocytes/retina, p<0.01). Furthermore, PSGL-1 blockade suppressed retinal RNA expression of TNF-α by 36% at 6h and by 35% at 24h after LPS, p<0.01 and p<0.05 respectively. Similarly, MCP-1 RNA expression was reduced by 25%, p<0.05 at 6h after LPS.

Conclusions:: The reduction of leukocyte rolling velocity on P-selectin provides direct evidence for functional upregulation of PSGL-1 on leukocytes during EIU. PSGL-1 blockade suppresses inflammation in experimental uveitis and may thus be a novel target for treatment of ocular inflammation.

Keywords: uveitis-clinical/animal model • retina • inflammation